Early Detection of Bacterial Infection in Acute Febrile Illness: How do We Use

Markers of Infection?
DEWI DIAN SUKMAWATI
Tropical and Infectious Diseases Division of Internal Medicine Department.
Sanglah Teaching Hospital Faculty of Medicine, Udayana University
Acute febrile illness defined as non specific terms for any illness of sudden onset
accompanied by fever. The fever, elevation in core body temperature above the daily
range for an individual, can be a characteristic feature of most infection: either bacterial,
viral, fungal or protozoan; and also found in a number of noninfectious diseases such as
autoimmune or auto-inflammatory diseases. In the process of making a diagnosis,
clinical history and physical examination has been the cornerstone of teaching medical
student, navigator and analytical foundation in diagnostic process. A good clinical skill
protects patients from unnecessary investigations with the risk of false positive results
and clinical risks that these investigations entail. Diagnostic study including laboratory
evaluation will add to focus diagnosis.
An ideal marker for bacterial infections should allow an early diagnosis, give
insight into the course and prognosis of a disease and able to facilitate the therapeutic
decision. Despite the advanced development in laboratory and diagnostic study,
currently no ideal markers were available for diagnosis of bacterial infection. The
diagnosis of bacterial infection will continue require critical clinical skills, careful history
taking, physical examination and culture for diagnosis confirmation.

THE COMPLETE BLOOD COUNTS (CBCS) AND INFECTION

White Blood Cells (WBC)
1. Neutrophilia
The myeloid line cells in bone marrow can be divided into: the mitotic pool
(blast to myelocytes) where the proliferation and maturation take place; the
maturation pool which end up as mature neutrophils; and the storage pool of
mature neutrophils and band form in marrow.
The presence of Neutrophilia in early response for bacterial infection is
due to mobilization of neutrophils in storage pool into the circulation.
Metamyelocytes are not released into circulation, except under extreme
circumstances. The Neutrophilia response can be blunted, for example the
present of underlying bone marrow disorder resulted in decreased capacity of

storage pool size thus the capacity to develop Neutrophilia in response for
bacterial infection is impaired.
Infection by Gram positive bacterial, usually present with Neutrophilia due
to increased of activated complement products, G-CSF, and pro-inflammatory
cytokines TNF-, IL-1 and IL-6. Some bacterial infection with insidious onset with
the present of splenomegaly (Typhoid fever, brucellosis) characteristically without
Neutrophilia, except in the beginning of disseminated phase. Neutrophilia in
chronic infection is maintained by increased in myeloid cells proliferation and
expansion of post-mitotic pool. Miliary tuberculosis should be one of differential
diagnosis for cases presenting with leukemoid reactions.
Infections due to Gram negative bacteria, especially the ones with septic
condition can cause either Neutrophilia or Neutropenia. Neutropenia occurs
when mature neutrophils being mobilized from marrow to circulation faster than
the proliferation. This condition picture the marrow with extreme shift to the left or
maturation arrest. Usually the Neutropenia in this setting accompanied with
thrombocytopenia and disturbance in coagulation profile. These changes depend
on the stage of sepsis, rate of peripheral consumption and bone marrow reserve.
The hematological disturbances are transient (may overshoot sometime before
settling) if we able to manage and control the septic condition. The first sign of
hematological improvements is often in normalization of thrombocytopenia.
2. Neutropenia
Neutropenia may be present in acute or chronic bacterial, viral, parasitic,
or rickettsial diseases. Infection due to exotoxin or endotoxin producing bacteria
(S.aureus, S.pyogenes, gram negative bacteria) frequently associated with
neutropenia or normal WBC with shift to the left (immature form) of WBCs. The
mechanisms are variable: direct damage to hematopoietic precursor cell
(infectious mononucleosis, hepatitis, HIV); bone marrow microenvironment
disturbance, especially stromal cell or fibroblast infection (CMV); infection of
endothelial cells (rickettsial, babesia) may cause pancytopenia due to
generalized vasculitis process; increased neutrophils adherence to damaged
endothelial cells (dengue, measles); increased mobilization and outstrip supply
(gram negative sepsis); splenic sequestration and marrow suppression (chronic
infection: TB, brucellosis, typhoid fever; malaria, kala-azar)
3. Lymphocytosis
If patient presents with fever and Lymphocytosis, the peripheral blood
smear is mandatory to differentiate between reactive Lymphocytosis or
lymphoproliferative disorder. Reactive Lymphocytosis may be seen in
mononucleosis syndrome (EBV, CMV, toxoplasmosis, viral hepatitis, HIV).
Pertussis due to Bordatella pertussis infection also present with marked

lymphocytosis with normal morphology which happened due to a delay in Thelper exit from blood into lymphoid tissue.
4. Lymphocytopenia
Lymphocytopenia typically be seen in primary or secondary
immunodeficiency (HIV infection, prolonged steroid or immunosuppressant drugs
use, malignancy). Mild to moderate lymphopenia also common in acute sepsis,
the condition related with glucocorticoid release. Acute viral infections may cause
lymphopenia due to direct destruction, trapping in spleen or lymph nodes, or
migration in regional / local site of viral infection (e.g. respiratory tract in
influenza).
Neutrophils to lymphocytes count ratio (NLCR) can be used to measure
the degree of systemic inflammation and predicting bacteriemia in emergency
setting. The most interesting study show the potential role of NLCR in
differentiate bacterial from viral infection which the cutoff value of 6.2 exhibit
sensitivity of 0.91 and specificity of 0.96 for bacterial infection.
5. Monocytosis
During acute bacterial infection, monocytes may increase as part of
leukocytosis, therefore the Monocytosis more significant in cases when total
WBC not increases. One fifth patients with subacute bacterial endocarditis
presented with Monocytosis, the increase of monocytes also be found in certain
viral infection (CMV), Monocytosis can be seen in resolution or convalescent
phase of acute infection.
6. Eosinophilia
Reactive Eosinophilia occurs when bone marrow proliferation of
eosinophils increase, induced by factors originating from T-lymphocytes
(cytokines IL-3, IL-5 produced in Th2 and for lesser degree in Th-1 response).
Typical infections with Eosinophilia include parasitic infection and fungal
diseases. Eosinophilia in tuberculosis or cryptococcal infection related with Th-2
response and often associated with poorly controlled infections.

Red Blood Cells (RBC): Anemia in Infection

Anemia commonly occurs in infection which last longer than a month and
considered as an anemia of chronic diseases (ACD). ACD is principally mediated by
TNF- and IL-1 which result in: shortened RBCs survival, impaired marrow
erithropoiesis response (blunted EPO response to anemia, impaired response erythroid
progenitor to EPO, enhanced apoptosis of erythroid progenitor, down regulation of
growth factor receptors on erythroid progenitor) and impaired iron mobilization and
utilization.

Other cause of anemia in infection including immune hemolytic anemia

(mycoplasma pneumoniae infection), non-immune hemolysis (malaria infection),
microangiopathic hemolysis (E. coli 0157), hemophagocytosis, direct infection of
progenitor cells (Parvovirus B19, HIV), iron deficiency (chronic bleeding due to H.pylorii
and peptic ulcer), and side effect of medication (antibiotic) use.
Thrombocytopenia and thrombocytosis
Thrombocytopenia in bacterial infection is usually due to peripheral causes
(consumptive coagulopathy and peripheral sequestration in splenomegaly).
Thrombocytosis that is reactive to the chronic inflammatory process may be
encountered in tuberculosis.
Thrombocytopenia in viral infections appears in two main settings, first as a almost
feature of disseminated infection in the neonate or immunosuppressed patient and
second as a complication of acute transient infection in previously healthy subjects.
There mechanisms of thrombocytopenia may be either immune mediated platelet
destruction with or without immune mediated megakaryocytes damage, or alternatively
direct toxicity from viral infection to megakaryocytes.

DISSEMINATED INTRAVASCULAR COAGULATION (DIC) AND SEPSIS

Septicemia is often associated with DIC, in particular infection by gram negative
bacteria (meningococcal infection). The triggering mechanism is due to initiation of
coagulation by endotoxin / bacterial lipopolysaccharide. Endotoxin activates factor XII to
XIIa, induces a platelet release reaction, cause endothelial sloughing with later
activation of XII-XIIa or XI-XIa, and release granulocyte procoagulant materials. Some
infections from Gram-positive bacteria are also noted to cause DIC through similar
mechanisms. Viremias are associated with DIC, the most common being varicella,
hepatitis, CMV and HIV infection.
DIC defined as systemic thrombohemorrhagic disorder with the evidence of
procoagulant activation, fibrinolytic activation, inhibitor consumption, biochemical
evidence of end organ damage or failure. The common laboratory abnormality
including: thrombocytopenia, increased D-dimer, prolonged PT, prolonged APTT,
prolonged thrombin time and reduced fibrinogen level. Blood smear shows
microangiopathic feature with fragmented RBCs (schistocytes), polychromasia,
spherocytes and thrombocytopenia. The differential diagnosis will be TTP/HUS which
also can be triggered by infection. The morphology from blood film is similar but the
coagulation profile in TTP/HUS usually normal.

ACUTE PHASE PROTEINS

Acute phase proteins are proteins whose levels are fluctuating in response to
tissue injury, either acute infection, burns, trauma, myocardial infarction or chronic
inflammation (malignancy, rheumatoid arthritis, Crohns disease); therefore this acutephase response is general and non-specific. The measurement can only be interpreted
based on clinical features case per case. The trigger for production is pro-inflammatory
cytokines such as IL-1, IL-6 and TNF-. Acute phase proteins including: C-reactive
protein (CRP), alpha-1 acid glycoproteins, alpha-1 antitrypsin, haptoglobin,
ceruloplasmin, serum amyloid A, fibrinogen, ferritin and complement component C3,
C4.
1. Erythrocyte sedimentation rate (ESR)
ESR is an indirect index of acute phase proteins concentration and depends on
fibrinogen concentration, a sensitive but non-specific index of plasma protein
changes in response to inflammation or tissue damage. ESR measures the rate
of RBCs sedimentation through a column of blood.
ESR affected by many factors: Haematocrit (HCT) variation, RBCs
abnormalities (ex. Sickle cells), delay in analysis (lysis of sample, therefore
should be analyzed within 4 hours), age, sex, menstrual cycle, pregnancy and
drug use (steroid). Normal ESR does not exclude organic disease. High ESR can
present in any inflammatory disorder (infection, RA), tuberculosis, early response
to myocardial infarct and anemia. Low ESR indicates polycitemia,
hypofibrinogenemia, congestive heart failure, spherocytosis, or sickle cells
diseases.
2. C-reactive protein (CRP)
Though non-specific (CRP also increases in inflammatory disorders,
tissue injury or necrosis, connective tissue diseases and malignancies beside in
infections), elevation of CRP in bacterial infection generally higher compared to
viral infections. In short term evaluation, CRP monitoring is better than ESR since
its level not depend on fibrinogen or immunoglobulin levels and not affected by
RBCs number and morphology.
CRP level will return to normal range after seven days of appropriately
and successful treatment for bacterial infection without concomitant complication.
Since CRP is not specific, the role in diagnosis is limited and better be used in
monitoring disease progression or activity (infection, malignancies, RA) or as a
prognostic marker (in acute pancreatitis).

3. Serum Amyloid A (SAA)

Serum amyloid A (SAA) proteins consist of a family of apolipoproteins
predominantly associated with high density lipoprotein (HDL) plasma. Their
expression consecutively elevated during inflammation stimuli. SAA synthesis
primarily in liver and the expression for some degree can be traced in extra
hepatic tissue. SAA is known best for its role as acute phase response to
infection stimuli, tissue injury and trauma. During acute inflammation, the SAA in
plasma can increase up to 1000 fold within 24 hours.
SAA is more sensitive (compared with CRP) but also as CRP, not specific.
SAA also cannot be used to differentiate infection due to bacterial or viral
infection.

PRO-INFLAMMATORY CYTOKINES (TNF-, IL-1, IL-6)

Cytokines are regulators of host responses to infection, immune responses,
inflammation, and trauma. Some cytokines act in amplification inflammatory reactions
(proinflammatory), whereas others serve to reduce inflammation and promote healing
(anti-inflammatory). Interleukin IL-1, IL-6 and tumor necrosis factor alpha (TNF-) are
proinflammatory cytokines, and during acute inflammatory response they produce fever,
inflammation, tissue destruction, and, in some cases, shock and death.
Studies failed to show consistent result in cytokines role on bacterial infection or
in their ability to differentiate between bacterial and viral infection. The inconsistency
especially seen in studies on TNF-, although TNF- is an evanescence cytokine
whose levels peak early and decline rapidly during sepsis. Alike most markers of
infection mentioned above, the pro-inflammatory markers also not specific which
elevated in several other conditions which initiate inflammatory response beside
infection.
PROCALCITONIN (PCT)
Procalcitonin is a 116 precursor peptide (pro-hormone) from the hormone
calcitonin (CT), after translation from CT-mRNA, PCT is cleaved enzymatically into
smaller peptides to yield 32 amino acid mature CT. CT is produced exclusively by Ccells in thyroid gland, in response to hormonal stimuli whereas PCT can be produced by
several other cells types in response to inflammation or infection. Since its identification
and association with sepsis in 1990s, a large number of studies involving PCT and its
clinical application have been conducted. PCT normally can be found in serum, in

bacterial infection and various inflammatory processes, the PCT level increase up to
several thousand folds. This increase and especially the course correlate with severity
of disease and mortality.
PCT is stimulated by bacterial products (endotoxin, lipopolysaccharide/LPS) and
cytokines (IL-1, IL-2, IL-6, TNF-). Plasma PCT has a half life of 2530 hours; the levels
typically increase within 36 hours of the stimulus. Higher levels are associated with
poorer prognosis. Elevated values are highly suggestive of an infection, typically
bacterial, with a systemic response (sepsis, or severe sepsis or septic shock). In healthy
people, PCT concentrations are typically below 0.05 ng/mL (see table), concentrations
can increase up to 1000 ng/mL in patients with sepsis, severe sepsis or septic shock.
Average levels of PCT increase from SIRS to septic shock, with the highest levels
associated with severe sepsis and septic shock. PCT has been shown to be a useful
tool that can aid in the diagnosis of sepsis. PCT levels can be helpful in facilitating early
diagnosis thus allowing for early therapeutic interventions that have been shown to
improve patient outcomes.

Table: Procalcitonin levels and potential causes

PCT (ng/mL)
<0.05

0.5 <2.0

2.0 <10

10

Possible causes
Normal value
Local inflammation or infection is possible, SIRS unlikely
On first day admission in ICU, indicates a low risk for progression to
severe sepsis and or septic shock
SIRS due to infection, or severe trauma, or major surgery, or
cardiogenic shock
If the patient has proven infection, it could be sepsis
Likely to be sepsis or severe systemic inflammation response due to
infection
On first day admission in ICU, indicates high risk for progression to
severe sepsis and or septic shock
Severe sepsis or septic shock
Organ dysfunction
High risk of death

The PCT optimal cutoff ranges are variable and depend on: the clinical setting
(primary care, emergency room, ICU, post operative or trauma patients), the site and

extent of infection, presence of co-morbidity or immunosuppression and clinical

implication setting (diagnostic, prognostic, antibiotic stewardship). False positive results
can be seen in condition: newborns, ARDS, acute plasmodium falciparum malaria
infection, systemic fungal infection, severe mechanical trauma, following surgical
trauma, admission monoclonal or polyclonal anti-thymocyte globulin for treatment of
transplant rejection, chemical pneumonitis, severe burns and heat stroke, patients with
medullary thyroid cancers, small cell carcinoma of the lung, carcinoids, tumor with
paraneoplastic hormone production, and inflammation associated with cytokines
storm.
False negative results can be found in early course of infection, localized
bacterial infection, and subacute bacterial endocarditis. Other limitation and weakness
of procalcitonin including: PCT is not very early marker of bacterial infection (follow up
and re-evaluation in suspicion of bacterial infection is important), single PCT value is not
a good prognostic marker, cost higher compared to CRP and different assay yields
different test performance.
SUMMARY
An ideal marker for bacterial infections should allow an early diagnosis and
should inform about the course and prognosis of the disease. Beyond any doubt, the
diagnosis of infections will continue to require a critical clinical awareness, careful
patient history, dedicated physical examination, and appropriate cultures in all patients.
However, the interpretation of the clinical response to a bacterial infection lacks
standardization and validation and is, therefore, prone to inter-observer variability. The
routine use of traditional markers of inflammation (eg WBC, CRP) and proinflammatory cytokines have inconsistent data on diagnostic accuracy and lack in
specificity but still can be use in conjunction with good clinical skills. PCT, even not a
perfect marker for bacterial infection, show promising role in diagnostic, prognostic and
antibiotic stewardship.

REFERRENCES
1. de Jager C. et al. The neutrophil-lymphocyte count ratio in patients with
community
acquired
pneumonia. PLoS
ONE. 2012;7:e46561.
doi:
10.1371/journal.pone.0046561
2. Holub M, Beran O, Kasprikova N, Chalupa P. Neutrophil to Lymphocyte count
ratio as a biomarker of bacterial infection. Cent. Eur.J.Med.2011

3. Urbonas V, Eidukait A, and Tamulien I, The diagnostic value of interleukin-6

and interleukin-8 for early prediction of bacteremia and sepsis in children with
febrile neutropenia and cancer, Journal of Pediatric Hematology/Oncology, vol.
34, no. 2, pp. 122127, 2012.
4. De Pablo R, Monserrat J., Reyes E., Mortality in patients with septic shock
correlates with anti-inflammatory but not proinflammatory immunomodulatory
molecules, Journal of Intensive Care Medicine, vol. 26, no. 2, pp. 125132, 2011
5. Charles P. E., Tinel C., Barbar S, Procalcitonin kinetics within the first days of
sepsis: relationship with the appropriateness of antibiotic therapy and the
outcome, Critical Care, vol. 13, no. 2, article R38, 2009