1. Indian Journal of Pediatrics, Volume 74—July, 2007 673
Correspondence and Reprint requests : Prof. B.D. Bhatia,
Department of Pediatrics, Institute of Medical Sciences, Banaras
Hindu University, Varanasi - 221005, India
[Received June 20, 2007; Accepted June 20, 2007]
Symposium : Newer Diagnostic Tests
Evaluation of patients with the signs and symptoms of
bacterial sepsis requires a combination of clinical acumen
and laboratory support. The diagnosis of bacterial
infection can be particularly difficult in certain patients,
such as neonates and young infants. Blood culture is
widely used for the diagnosis of septicemia.However, in
many clinical situations the yield from blood culture is
low, positive cultures are obtained from fewer than 30%.
Some of the patients with false-negative blood culture
may have had prior antibiotic treatment or they were not
bacteremic at the time of blood collection. Consequently,
considerable effort has been devoted to the development
of rapid, sensitive and specific assays for the detection of
causative organisms. In the last few years many new
techniques have entered into the field of early diagnostic
process of bacterial infections which we have subdivided
further into three broad headings as described below:
I. Direct demonstration of bacteria by identifying
bacterial genomes
II. Demonstration of indirect evidences of bacterial
infection by identifying serological markers of
systemic inflammatory response syndrome
III. Rapid bacterial culture methods
I. DEMONSTRATION OF BACTERIA BY
IDENTIFYING BACTERIAL GENOMES
During the past decade, there has been unprecedented
progress in molecular biology as well as in the application
of nucleic acid technology to the study of the
epidemiology of human infection, some of which are
summarized below.
1. Polymerase Chain Reaction: Today, the most widely
used nucleic acid amplification and detection method, the
Polymerase Chain Reaction (PCR) assay, has been found
to have a substantial impact on the diagnosis of infectious
diseases.1,2
The ability of this method to amplify minute
amounts (less than 3 copies) of specific microbial DNA
sequences in a background mixture of host DNA makes it
a powerful diagnostic tool.3
Nucleic acid amplification is
performed in a thermocycler, which is an instrument that
can hold the assay’s reagents and allows the reactions to
occur at the various temperatures required. In the initial
step of the procedure, nucleic acid (e.g., DNA)is extracted
from the microorganism or clinical specimen of interest.
Heat (90ºC-95ºC) is used to separate the extracted double-
stranded DNA into single strands (denaturation). Cooling
to 55ºC then allows primers specifically designed to flank
the target nucleic acid sequence to adhere to the target
Newer Diagnostic Tests for Bacterial Diseases
B.D. Bhatia and Sriparna Basu
Department of Pediatrics, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India
ABSTRACT
In diagnosing bacterial infections, the rapid identification of bacteremia at an early stage of the disease is critical for a favorable
outcome. Furthermore, it is important that exact information be obtained on the stage of the disease rapidly in order to choose
and initiate the appropriate therapy. In recent years many new techniques have been added in the diagnostic tools. During the
past decade, there has been unprecedented progress in molecular biology as well as in the application of nucleic acid technology
to the study of the epidemiology of human infection. Highly sensitive molecular techniques are found to be capable of detecting
minute amounts of specific microbial DNA sequences and their complex genetic variations. Moreover, altered levels of
biomarkers such as procalcitonin, C-reactive protein, tumor necrosis factor alpha, and several interleukins are also found to be
promising to define systemic inflammatory response syndrome as indirect evidences of bacterial infections. Lastly, many rapid
culture methods are coming up to achieve faster bacterial diagnosis. In this review we will focus on these three newer methods
for the early diagnosis of bacterial infections. These approaches will help to expedite the diagnosis of especially early infections
and might be a further step towards the improvement of therapeutic methods. [Indian J Pediatr 2007; 74 (7) : 673-677]
E-mail : baldev_bhatia@rediffmail.com
Key words : Bacterial infections; Sepsis; Polymerase chain reaction; Proinflammatory mediators; Culture
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2. B.D. Bhatia and S. Basu
674 Indian Journal of Pediatrics, Volume 74—July, 2007
DNA (annealing). Following this, the enzyme Taq
polymerase and nucleotides are added to create new
DNA fragments complementary to the target DNA
(extension).This completes one cycle of PCR. This process
of denaturation, annealing and extension is repeated
numerous times in the thermocycler. At the end of each
cycle each newly synthesized DNA sequence acts as a
new target for the next cycle, so that after 30 cycles
millions of copies of the original target DNA are created.
The result is the accumulation of a specific PCR product
with sequences located between the 2 flanking primers.4
A major advantage of PCR is that it is far more sensitive
than direct hybridization and thus requires extremely
small amounts of target DNA as a template. PCR-based
methods can detect as few as 10 to 100 copies of bacterial
genome in clinical samples. The sample does not have to
be highly purified, and even the starting DNA may be
partially degraded as long as the target sequence (~100–
1000 bp) is intact. The rapidity of PCR makes it possible
to generate detectable amounts of amplified target
nucleic acid within hours, compared with several days by
probe hybridization procedures and often weeks for the
identification of many fastidious organisms by culture.
Conventionally, the sequence of the 16S rRNA gene has
been used to detect and identify bacterial infection in
clinical practice. The concept of DNA microarray
hybridization is introduced recently, and the technique
has been applied in clinical diagnosis and many fields of
research, e.g., functional genomics and genetic analysis.
Microarrays are usually coupled with PCR where they
serve as a set of parallel dot-blots to enhance products
detection and identification.5
PCR assays that are currently available commercially
for use in diagnostic laboratories include tests for the
detection of Chlamydia trachomatis, C. pneumoniae,
Mycobacterium tuberculosis, Mycoplasma pneumoniae and
Neisseria gonorrhoeae.6
Multiplex PCR-based assays have
been developed and have the advantage of detecting
multiple pathogens in a single PCRreaction. These have
been used to detect common bacterial causes of
respiratory tract infections, bacteremia and meningitis.
Another promising application of PCR is in the diagnosis
of infectious conditions in which there are often too few
organisms present for detection by other means (For
example, for Borrelia burgdorferi in Lyme disease, the
range of symptoms and the limited diagnostic methods
available frequently delay the diagnosis).7
Assays have also been developed that can detect
genomic sequences associated with antimicrobial
resistance. PCR-based methods for the detection of
antimicrobial resistance have been applied to bacteria
including methicillin-resistant Staphylococcus aureus,
vancomycin-resistant enterococci and multidrug-resistant
M. tuberculosis.
Despite the obvious advantages to these newer
procedures, there may be potential limitations to DNA
amplification technology in the diagnostic microbiology
laboratory. The accuracy and reproducibility of PCR
assays depend on the technical expertise and experience
of the operator. The extreme sensitivity of PCR
paradoxically leads to one of its major drawbacks—the
occurrence of false-positive results. Very small amounts
of the amplified target sequence, of which up to 10 copies
can be present in a single PCR solution, can contaminate
laboratory equipment or reagents. The PCR product can
even spread as airborne droplets in areas of sample or
reagent preparation. This contaminating DNA can then
serve as a template for further amplification, resulting in
false-positive results in subsequent samples. For this
reason laboratories should have separate rooms for
different steps of the PCR procedure and must follow
stringent quality control measures to prevent
contamination or carry-over. False-negative test results
may occur because of the presence of substances in the
specimen that inhibit nucleic acid extraction or
amplification. Certain specimen types (e.g., blood) are
more likely to contain such inhibitors. The assays may
also lack sensitivity if there is a low inoculum of the
microorganism present in the clinical specimen. This may
be exacerbated if an inadequate sample or very small
specimen volume (i.e., < 20 µL) is available for testing.
Another source of error is the detection of nonviable
organisms by PCR, since it amplifies only a portion of the
microbe’s genome. In such instances, the detection of
complementary DNA by reverse-transcription PCR of
messenger RNA encoded by the pathogenic organism
can serve as evidence of active infection. Interpretation of
nucleic acid amplification test results is not always clear-
cut. For example, assays may detect the residual DNA of
a pathogenic microorganism even after successful
treatment, and it is not clear whether this represents the
presence of a small number of viable organisms or
amplified DNA from nonviable organisms. Therefore,
PCR tests should not be used to monitorthe effectiveness
of a course of therapy. Finally, it must be acknowledged
that performance of a PCR assay is generally more
expensive than conventional diagnostic laboratory
methods.
Nested PCR is a variation of the polymerase chain
reaction (PCR), in that two pairs (instead of one pair) of
PCR primers are used to amplify a fragment. The first
pair of PCR primers amplify a fragment similar to a
standard PCR. However, a second pair of primers called
nested primers (as they lie / are nested within the first
fragment) bind inside the first PCR product fragment to
allow amplification of a second PCR product which is
shorter than the first one. The advantage of nested PCR is
that if the wrong PCR fragment was amplified, the
probability is quite low that the region would be
amplified a second time by the second set of primers.
Thus, Nested PCR is a very specific PCR amplification.
~
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3. Newer Diagnostic Tests for Bacterial Diseases
Indian Journal of Pediatrics, Volume 74—July, 2007 675
2. Nucleic Acid Hybridization and Restriction Fragment
Length Polymorphism Analysis: Nucleic acid
hybridization in its simplest form can be used in the
detection of microorganisms or specific resistance genes.
It confirms the results of microbial cultures or even
detects organisms in clinical samples. This may require
the extraction of DNA or, in some cases, RNA from a
clinical sample (body fluid, peripheral white cells,
aspirate or scraping, or fresh tissue). A labeled synthetic
nucleic acid probe often less than 30 bases long can detect
the presence of target nucleic acids (oligonucleotide
probe). The oligonucleotide probes directly identify
microbial genetic sequences in contrast to conventional
immunologic tests and Western blot techniques, which
pick up microbial gene products or proteins. Molecular
hybridization has greater sensitivity and specificity than
these conventional techniques.
3. RNA Typing or Ribotyping: RNA typing or ribotyping
is another chromosomal detection technique. There is
conservation of the genes that encode subunits of
ribosomal RNA between species. Ribosomal genes are
present throughout the chromosomes of bacteria: the
sequences of DNA between the ribosomal genes vary in
length. The digestion of chromosomal DNA by
endonuclease produces random fragment polymorphic
patterns when probed with ribosomal RNA. For this
method, E coli 16S ribosomal RNA serves as a probe for
the endonuclease-digested chromosomal DNA. Because
chromosomal nucleotide patterns usually vary from
strain to strain but not within a strain, this technique can
identify organisms and differentiate strains. The
technique has application in the typing of Haemophilus
influenza, Pseudomonas capacia, E coli, Salmonella typhi, and
Providencia stuartii.8
However, ribotyping is not as useful
for gram-positive organisms; for instance, in enterococci,
it does a little more than differentiate to a species level.
4. PCR Ribotyping: PCR ribotyping is the analysis of
banding patterns obtained by gel electrophoresis of PCR-
amplified fragments of the 16S to 23S ribosomal RNA
intergenic spacer regions. It has considerable advantages
in terms of speed and technical ease, and is useful in
detecting and typing C difficile strains.9
5. Pulsed-Field Gel Electrophoresis: This is a widely
used technique for analyzing a large amount of
chromosomal DNA found in large bacterial chromosomal
fragments generated by endonuclease digestion.10
6. Clamped Homogeneous Field Electrophoresis:
Investigators have developed this method to compare
large chromosomal fragments generated by restriction
endonuclease digestion. This technique, a form of pulsed-
field gel electrophoresis, may be an easy way to compare
isolates of a species. Clamped homogeneous field
electrophoresis helps in the analysis of vancomycin-
resistant enterococci. No reliable typing system
previously has been able to identify strains of this
organism beyond the species level. This technique can
type other bacteria, mycobacteria, and yeast as well.
7. Multilocus Sequence Typing : In principle, this is the
genome-based version of the conventional method of
multilocus enzyme electrophoresis. It helps in the typing
of various bacterial species by identifying DNA alleles
from various organisms, including Campylobacter jejuni, S
aureus, and more recently, Enterococcus faecium. The
method involves PCR amplification and the nucleic acid
sequencing of multiple internal fragments of
housekeeping genes. The advantages of this approach are
that the culturing of pathogenic microorganisms is
avoided, as their gene fragments are amplified directly
from biologic samples, and that the sequencing data are
unambiguous, easy to standardize, and electronically
portable.
8. Fluorescence-Based Amplified Fragment Length
Polymorphism: This is a novel assay based on the
fluorescent analysis of an amplified subset of restriction
fragments. The fluorescence-based amplified fragment
length polymorphism assay involves the selective PCR
amplification of restriction fragments from a total digest
of genomic DNA. It has been useful in the study of
vancomycin-resistant enterococci.11
9. DNA Sequencing and Molecular Evolutionary
Analysis: The sequencing of DNA refers to the
enumeration of individual nucleotide base pairs along a
linear segment of DNA. Single-stranded or double-
stranded DNA generated by PCR can be used directly for
DNA sequencing. Automation has made it possible to
double the lengths of readable DNA sequences obtained
after a single sequencing run to more than 400 to 800 bp.
Recent studies of microbial populations have
demonstrated substantial genetic diversity within
microbial species on a very minute level. Organisms have
relatively small generation times. They have evolutionary
divergence, as with any other species. This is due to the
accumulation of random, nonlethal mutations. These may
include minute changes, including single base pair
substitutions, the deletion of individual genes, or even the
acquisition of DNA from other microbial species. Over
time, these lead to substantial genetic diversity within
microbial species and indicate that isolates of organisms
have many genetically diverging lineages. Highly
sensitive molecular techniques are capable of detecting
single base pair substitutions and resolving the
mechanism of underlying complex variation and
epidemiology.
II. INDIRECT EVIDENCES OF BACTERIAL
INFECTION BY IDENTIFYING SEROLOGICAL
MARKERS
In 1991, the American College of Chest Physicians and the
Society of Critical Care Medicine convened a Consensus
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4. B.D. Bhatia and S. Basu
676 Indian Journal of Pediatrics, Volume 74—July, 2007
Conference in an attempt to provide a conceptual and
practical framework todefine the systemic inflammatory
response to infection, which is a progressive injurious
process that falls under the generalized term sepsis.
Sepsis is considered when there is a systemic response to
a possible infection, termed as systemic inflammatory
response syndrome (SIRS). The quest for surrogate
biomarkers to define SIRS has identified several potential
candidates. Markers such as procalcitonin, C-reactive
protein (CRP), tumor necrosis factor alpha (TNF-α), and
several interleukins appeared promising in initial studies.
Several of them are described below.
1. Cytokines : As soon as a bacterium enters the body, it
is confronted with two lines of defense: a humoral line
and a cellular line. The humoral factors comprise
complement, antibodies, and acute-phase proteins. In the
cellular line of defense, in particular the mononuclear
cells (monocytes and macrophages) and the neutrophils
are of great significance since these cells may recognize
bacterial cell wall constituents directly or indirectly after
complement and antibody bind to the bacterium and its
constituents. Bacterial cell wall constituents such as
lipopolysaccharide (LPS), peptidoglycans, and
lipoteichoic acid are particularly responsible for the
deleterious effects of sepsis. These constituentsinteract in
the body with a large number of proteins and receptors,
and this interaction determines the eventual
inflammatory effect of the compounds. Within the
circulation bacterial constituents interact with proteins
such as plasma lipoproteins and lipopolysaccharide
binding protein. The interaction of the bacterial
constituentswith receptors on the surface of mononuclear
cells is mainly responsible for the induction of
proinflammatory mediators bythe bacterial constituents.
Recognition of LPS or other bacterial componentsinitiates
a cascade of release of inflammatory mediators, vascular
and physiological changes, and recruitment of immune
cells. An LPS-activated macrophage becomes
metabolically active and produces intracellular stores of
oxygen free radicals and other microbicidal agents
(lysozyme, cationic proteins, acid hydrolases, and
lactoferrin) and secretes inflammatory mediators like
TNF-α, IL-1, IL-2, IL-6, IL-8, IL-10, IL-12, IL 18, gamma
interferon (IFN-γ), and granulocyte-macrophage (GM)-
CSF, platelet-activating factor (PAF), chemokines,
eicosanoids and anaphylatoxins C3a and C5a.
Measurement of these cytokines can be taken as
fingerprints of sepsis. The host inflammatory responses to
gram-negative and gram-positive stimuli share some
common response elements but also exhibit distinct
patterns of cytokine appearance and leukocyte gene
expression. There are marked differences in the responses
to gram-positive and gram-negative bacteria. Whereas,
gram-negative bacteria all contain LPS as their major
pathogenic determinant, gram-positive bacteria contain a
number of immunogenic cell wall components besides
the highly deleterious exotoxins. Concentrations of tumor
necrosis factor alpha, interleukin 1 receptor antagonist
(IL-1Ra), IL-8, IL-10 and IL-18 binding protein in plasma
do not differ between patients with sepsis due to gram-
negative and gram-positive bacteria. However, plasma
IL-1β, IL-6, and IL-18 concentrations are significantly
higher in patients with sepsis due to gram-positive
bacteria. The immunological response to gram-negative
bacteria mainly involves leukocytesand the production of
cytokines such as tumor necrosis factor alpha (TNF-α),
interleukin-1 (IL-1), and IL-6. The release of exotoxins,
many of which are superantigens, by gram-positive
bacteria activates T cells, resulting in a different cellular
response and different cytokine profile, with relatively
lowlevels of TNF-α, IL-1, and IL-6 and increased levels of
IL-8.9
Several studies have shown elevated plasma IL-18
concentrations to be associated with poor clinical out-
come in severe inflammatory and septic conditions.12, 13
2. Procalcitonin (PCT): PCT is a relatively new marker
that has been associated with inflammation and sepsis. It
is a 116-amino-acid protein that is the precursor to
calcitonin. The PCT plasma level in healthy individuals is
low; usually below 0.1 ng/ml. The levels have been
shown to rise with severity of sepsis. The sensitivity of
PCT in initial determinations for the diagnosis of sepsis
has been reported to vary from 61% to 85%, increasing to
72–100% within the subsequent 24 h. PCT specificity was
found to vary in initial determinations from 50% to 97%
and ranged between 63% and 97% within the next 24 hr.14
PCT had the highest sensitivity and specificity for
differentiating SIRS from sepsis. It has been found to be a
more reliable marker in the diagnosis of sepsis than other
measures.
3. C-reactive protein (CRP): The acute phase protein CRP
is released from the liver in response to proinflammatory
cytokines and thought to recruit monocytes in early
infection. Sensitivity and specificity of CRP levels in older
children has been poor; however, in newborns CRP
specificity averages 90%. Maximum CRP levels >3 mg/
dL had positive predictive values >20% for proven or
probable early-onset infections and for proven or
probable and proven late-onset infections, but only those
>6 mg/dL had such a high positive predictive value for
proven early-onset sepsis. Serial CRP levels are useful in
the diagnostic evaluation of neonates with suspected
infection. Two CRP levels <1 mg/dL obtained24 hr apart,
8 to 48 hours after presentation, indicate that bacterial
infection is unlikely. The sensitivity of a normal CRP at
the initial evaluation is not sufficient to justify
withholding antibiotic therapy. The positive predictive
value of elevatedCRP levels is low, especially for culture-
proven early-onset infections.15
4. Cell surface receptors (neutrophil CD64 and CD11):
Neutrophil CD64 expression quantitation provides
improved diagnostic detection of infection/sepsis
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5. Newer Diagnostic Tests for Bacterial Diseases
Indian Journal of Pediatrics, Volume 74—July, 2007 677
compared with the standard diagnostic tests used in
current medical practice with a sensitivity of 87.9% and
specificity of 71.2%. Neutrophil CD64 is one of many
activation-related antigenic changes manifested by PMNs
during the normal pathophysiologic process of acute
inflammatory response. Neutrophil expression of CD64 is
upregulated under the influence of inflammation-related
cytokines such as interleukin 12, interferon gamma, and
granulocyte colony-stimulating factor. CD64 appears
ideal as a surrogate marker of PMN activation or of
systemic acute inflammatory response because its
expression begins at less than 2000 sites per cell and
becomes up-regulated in a graded fashion depending on
the intensity of cytokines stimulation. Furthermore, the
up-regulation of PMN CD64 expression appears to be of
pathophysiologic significance because this form of Fc
receptor has been shown to be functional in eliciting all
the PMN functional responses used in antibacterial
responses.16
Another cell surface receptor CD11 is also
found to be upregulated during infection.
III. RAPID CULTURE METHODS
1. BacT/Alert, BACTEC 660/730 and VITEK 2
nonradiometric blood culture systems: The detection of
bloodstream infections is one of the most important tasks
performed by the clinical microbiology laboratory. Rapid
bacterial identification and susceptibility testing not only
improve patient therapy and outcome, but also reduce
costs. Both automated blood culture systems and
automated systems for identification and susceptibility
testing of bacteria have been on the market for a number
of yr. These systems use fluorescence-based technology.
They are used widely due to the characteristics such as
lower contamination risks, shorter incubation periods
and higher isolation rates. Though both rapid and sen-
sitive, false-positive and false-negative results are high.17
2. The rapid evaluation of bacterial growth and
antibiotic susceptibility in blood cultures by selected
ion flow tube mass spectrometry: To achieve faster
bacterial diagnosis, selected ion flow tube mass
spectrometry (SIFT-MS) measured metabolic gases in the
headspaces of BacT/ALERT blood culture bottles.
Pseudomonas aeruginosa, Streptococcus pneumoniae,
Escherichia coli, Staphylococcus aureus and Neisseria
meningitidis growth and trace gas patterns were detected
from 10 colony forming units after 6 hours.18
In a recent study of ours (unpublished data) blood
culture was positive only in 24 out of 55 cases of neonatal
sepsis, whereas nested PCR was positive in 83.6% of
suspected cases of neonatal sepsis. Even the primary PCR
was positive only in 25 out of 55, whereas nested PCR
was positive in 21 out of 30 cases where primary PCR was
negative. In 15 suspects where all tests were negative,
nested PCR was positive in 12 cases.
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