Clinical Microbiology Lecture3

10/11/2021

Clinical Microbiology-Lecture 3
Clinical Microbiology-03
Bilinc Kavak and Melis Engin
10/11/2021
Rapid molecular diagnosis of the pathogen

Kary Mullis was an American biochemist. In recognition of his role in the invention of
the polymerase chain reaction (PCR) technique, he won the Nobel Prize in Chemistry.
Thanks to him we have the PCR technique in our labs today. Professor highly suggested we
read his autobiography.

Microorganisms in Blood

In general, the idea is that sepsis is generated by bloodstream infections is not

completely true. But 70% of the patients who suffer from sepsis also suffer from bloodstream
infections.
Bloodstream is one of the few anatomical body districts that is considered to be sterile.
The sentence “There should be no microorganism in the blood” is not completely true
because if you try to detect DNA fragments deriving from bacteria you can pick up some
sporadic fragments in healthy people. We can separate the bacteria present in blood can
into 3 different categories depending on the timing.

1) Accidental: We have a spike of infecting bacteria that move from a site of infection
(e.g., diverticula in the gut, apical granuloma in the teeth, etc.).
We have many different possibilities in which we have a very c of infection with the
multiplication of bacteria. Sometimes localized infections can burst suddenly which
leads the bacteria to move into the bloodstream. This is a transient situation that
doesn’t last long, mainly because of the low number of bacteria that can enter the
bloodstream. As the number of bacteria that enter the bloodstream is low, the
cellular response from the white blood cells (WBC) is capable of clearing
everything in a few seconds to minutes.

2) Intermittent bacteria presence: This is again generated by a larger localized site of

infections that open in the direction of the bloodstream with more frequent episodes
compared to the accidental finding of bacteria in the bloodstream. That means,
according to this, you can pick up the bacteria not so frequently, but it is possible to
find them in the bloodstream. This is a potentially pathological condition. WBCs can
clear them out.

3) Persistent bloodstream infection Once the bacteria enter the bloodstream with a
high bacterial load, they may not be cleared away and generate the bloodstream
infection.

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Sepsis Pathogen Identification

Nowadays the bloodstream infection represents one of the most relevant types of
infections both epidemiologically and clinically that we must deal with. It’s because the
mortality of the bloodstream infections that evolve into sepsis have high mortality. Sepsis
can evolve to septic shock which is devastating for the body as it is a shock condition and
we have total organ failure one after another (liver, kidney, heart, etc.).
The mortality rate for patients with septic shock is 70%. Even if you can treat the infection
that is generating this multi-organ failure (septic shock) with adequate antimicrobial and
antibacterial therapy, the cycle is self-maintaining and self-progressing towards shock.

We can observe 2 periods:

1. The first period in which you can efficiently treat the infection to stop the
progression of this catastrophic situation.
2. In the second period, you need to move your therapy in the direction of life-
supporting therapy and no more anti-infective therapy. At this point, the infection is
just a trigger that started the situation, but it is not very relevant as even if you cure
the infection the patient will progress very fast in this
dangerous self-maintaining cycle.

As we can see in the graph the blue curve is showing the

mortality rate per cent seen in the sepsis affected populations
that start from SIRS (systemic inflammatory response
syndrome) which is a pre-septic condition. Then we have multi-
sepsis, severe sepsis, and finally septic shock. Moving from
sepsis to septic shock, the mortality rate increases from 20% to
70%. On the other hand, we can also see the incidence in
thousand cases closed and localized sites worldwide of different
cases we just mentioned. Not many patients evolve to septic
shock. It’s important to mention again that this mortality rate is
very impressive because there is no other pathological condition
that can bring the mortality rate up to 70% in such a short time.

In the picture on the left, we can see how long it could take
for the microbiology lab to respond to the question “Does this
patient have any kind of microorganism that is generating
sepsis?”.
You start with collecting the blood culture and incubating it
in the automated system and in about 15 hours you get the
positive blood culture result. But this positive blood culture
result needs another 1.5 days for pathogen identification
results. You can get antimicrobial susceptibility results in an
additional half a day. In total this whole process can take up
to 3 days, but you don’t have 3 days available. The critical time
in which intervention can make a vast difference in the
prognosis of these patients is about 12 hours. That means that
anytime we go for classical workflow we are always late in
respect to the clinical needs of patients suffering from sepsis.
This causes problems between microbiologists and clinicians
as the clinicians want the test results as soon as. As blood
culture is negative before 15 hours microbiologists are unable
to give any results. We need to find a possible solution to this.

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Emerging Technologies for Molecular Diagnosis of Sepsis (slide 4)

In this paper published 3 years ago, we can see the required features of an ideal
diagnostic technology to impact the clinical management of a patient suffering from sepsis.
In this list of criteria, we can see that some of them are not fulfilled by present diagnostic
technologies in the laboratory.

1) Rapid testing/detection. The pathogen needs to be identified in less than 3 hours

because when we have a sepsis patient in the ER, there’s usually already a fever of
unknown origin lasting for 24-36 hours and mental confusion (so you can ask them
some basic questions like what day is today, etc. and you might not get a correct
response).
If you start the treatment of this patient with the right antibiotic therapy within 3 hours
of arrival in ER, you can stop the progression of sepsis. This is only possible if you
are a lucky doctor because you start with empirical antibiotic therapy as you don’t
know the specific pathogens involved in this patient. You can have more of a specific
idea if you have strong epidemiological support given by the clinical microbiology
laboratory. A clinical microbiology laboratory can give you an idea about what is the
most hypothetically frequent aetiology of the sepsis patient you see in your clinical
setting. In western countries and Southern Europe, the most frequent aetiology is
Gram-negative bacteria of the family of Enterobacteriaceae (E. coli, Klebsiella,
etc.). Whereas in Northern Europe the most frequent bacteria are Gram-positive
bacteria.
So, according to this information you can try to set up an empirical therapy that
makes sense. This information should be locally and timely updated as they can
differ for example from Bologna to Milan. It makes no sense to work on
epidemiological data that is older than 6 months. Ideally, you should get information
monthly.

2) You should be able to provide the broad basic detection including bacteria, viruses,
and potentially fungi. You never know the causative pathogen of your sepsis patient
in the beginning. So, the larger the panel of pathogen potentially identified, the more
impacting results we get.

3) You should be able to provide all this information with a very limited amount of blood.
Of course, normally you can withdraw 50ml of blood, but sepsis patients are patients
that are already in a very unstable equilibrium under the hemodynamic point of view.
So, you cannot just withdraw any amount of blood you want. You are allowed to
withdraw the amount of blood that is necessary which is less than 1ml for pediatric
patients and neonates; 5-10ml blood for adults.

4) These tests should have high sensitivity and specificity for the immediate initiation of
targeted antibiotic use in the presence of signs and symptoms of systemic
inflammation.

5) It should be also possible to identify polymicrobial detection of pathogens. In general,

by using the blood culture techniques, it is quite difficult to find polymicrobial
infection. Because if you have a couple of bacteria in your patient’s blood, the
individual bacterial load for each species couldn’t have been the same. For example,
you can have a very high concentration of E. coli and a very low concentration of
Staphylococcus aureus. The possibility to identify the polymicrobial infection is highly
influenced by the speed of growth. Because, if the amount of E. coli gets doubled
every 30 minutes which is very fast, even if you have a low E. coli bacterial load at

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the beginning you will get a huge amount in a very short time. And if you have a
bacterium that grows slower than E. coli, E. coli would be the predominant species in
your sample, and you will just pick up the fastest-growing bacteria. So, detecting
polymicrobial infection is very relevant because sometimes you treat for one
aetiology, but it is more than 1 germ involved.

6) It should also be able to detect drug resistance. Of course, antibiotic therapy should
be driven by epidemiological data about antimicrobial resistance but if you can get
precise information about the antimicrobial resistance related to the germ that has
been isolated from your patient, it would be the best scenario for the patient.

7) The best possible way would be to have a test that can be located near the bed of
the septic patient. So, this way you can reduce the logistic time and turnaround time
of your test. In general, the laboratories are not located inside the hospitals anymore.
In the hospital, we can find small urgent testing laboratories that could run the test
24/7. But we don’t have the availability of large laboratories near the hospitals as it is
very expensive to have one large laboratory serving only one hospital. It is not
sustainable. So, we need to find a sustainable solution to have large laboratories that
can run urgent tests inside the hospitals.

Management of Septic Patients Clinically

Look at the different periods bottom of the graph. One is the period so-called blood
culture, in the green on the graph on the far-right side you see the conventional identification
and susceptibility testing. As we can see from the timeline on the bottom of this graph, it
reports an average of 12 hours to get a positive blood culture result and an additional 24
hours (so 36 hours overall) for the final identification of the germs inside the blood culture.

Let’s move to the upper side of the picture. We can see 2 identical bars, but the first
one is empirical antibacterial treatment as you don’t have any information. So, at this time
you can only treat the patient according to epidemiological data available. Again, the more

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accurate the data, the more accurate treatment you can provide to this patient. Following this
first empirical antimicrobial treatment period, we will find the adjustment/de-escalation period
of therapy.
Adjustment means that changing the complexity of your empirical treatment
according to the reports you get from the laboratory. For example, if you have started your
empirical treatment with drugs against Gram- bacteria like E. coli, but after a while, if the lab
reports find out that there is also Staphylococcus aureus which is Gram+, you need to add
drugs that are effective against Staphylococcus aureus and adjust your treatment.
De-escalation is a mechanism whereby the provision of effective initial antibiotic
treatment is achieved while avoiding unnecessary antibiotic use that would promote the
development of resistance. It is a key element within antimicrobial stewardship programs
and treatment paradigms for serious sepsis. The embodiment of de-escalation is based on
the microbiology results around the day 3 therapy point; the empiric antibiotic(s) that were
started, are stopped or reduced in number and/or narrowed in the spectrum. Let’s say you
started with combination therapy with at least 2 different drugs for both Gram+ and Gram-
bacteria. However, after the lab report states that there are only Gram+ bacteria, you should
stop the drug for Gram- bacteria. As also every antimicrobial drug has side effects
(damaging of gut microbiota, kidney failure, etc.), you should prevent the patient from the
unnecessary toxicity of the drug. But in practice, this is rarely done as most of the time
doctors don’t trust the microbiology laboratory. This happens especially in ICU as doctors try
to give every possible therapy to stabilize the sepsis patient. When microbiologists ask ICU
doctors whether they modified the treatment according to the lab reports they received,
almost always the answer is “they are my patient!” from the ICU doctors. But for the reasons
mentioned above (toxicity and antibiotic resistance) de-escalation should be deeply
enforced.

Let’s move to the middle of the picture. We can see all the possible diagnostic
techniques that you can apply to each one of these divided periods of your septic patient. In
the first part, you can make a diagnosis from primary whole blood. That means you should
not go for any culture-based technique on blood. You just go for the direct detection of the
pathogen in the primary whole blood. If this works, you can get very precise information in
less than 12 hours. That means if you can apply these kinds of technologies, you can get the
information you need faster than the time that is usually required to have just a positive
blood culture. It is a reduction in the turnaround time of your workflow for more than 24
hours. That means you can act in the adequate time and in the right way in the so-called
golden hours of the septic patient.

When we look at the right middle half of the picture, we can see a lot of different
technologies. You need to wait 12 hours for a positive blood culture. After getting a positive
blood culture result, we have these different technologies for further testing.

You can start your diagnostic work with a very old technique called Gram-staining
technique once you have a positive blood culture. You need to open the blood culture, make
a smear, stain the smear with gram staining and take this slide under the microscope to find
out if there are Gram+, Gram- bacteria, and the shape of the bacteria you are observing.
This is extremely informative. Of course, it doesn’t provide the clinical report with the name
of the genus of the species or antimicrobial susceptibility testing, but it provides a general
direction for the treatment when combined with local epidemiological data. Let’s say after
you see a series of Gram+ cocci in the shape of grapes you can conclude that you are
dealing with Staphylococcus aureus. This Gram staining can be done just after 15 minutes of
getting a positive blood culture. So overall it can orient the therapeutic approach very
precisely.

Then you have MALDI-TOF which is a molecular technique. It is mass spectrometry.

We also have FISH, microarrays. We can also have combination tests like POCT-PCR. All

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these techniques have limitations. The biggest limitation for all is that you are pre-selecting
the germs that you want to know. Anytime you apply something to PCR, something related
to DNA amplification, you need to have a set of primary probes that means you just amplify
what you want to amplify.

Turnaround time after positive blood

culture depends on the technique used. It is 1.5-3
hours(h) for hybridization techniques. 1-8 h for
amplification techniques and 4-6 h for mass
spectrometry. Turnaround time after the whole
blood sample is just 12-24 hours, in addition to
each method’s turnaround time after positive
blood culture. Turnaround time after the whole
blood sample is at least 6 hours (in practice 6
hours is very optimistic but it is possible on the
same day of the sampling of the blood) for whole blood amplification methods directly from
the blood sample.

Question: At the beginning of the lecture, we said that we have the presence of bacteria in
the bloodstream in different ways (accidental, intermittent, and persistent). Are we talking
about a blood vessel rupture so in this way bacteria enters blood?
Answer: In the case of diverticula, we are talking about absorption. Bacteria can enter the
mucosa or submucosal layer in the gut and from there they can move to the bloodstream.
But also, sometimes we can have intermittent or transient bacteremia deriving from rupture
of the integrity of the tissue, like when you brush your teeth very forcefully you can generate
some hemorrhagic leakage from the gums, and this is just an open door that leads bacteria
that are in the periodontal space or oral cavity to enter the blood. Bacteremia can also be
caused by some iatrogenic endoscopic procedures.

Question: We said that we attenuate some antimicrobial therapies as they have side
effects. Do we also attenuate when we have ineffectiveness of the drug or when the patient
is not responding to the treatment?
Answer: Clinical responsiveness of the patient is the basic point to evaluate the efficacy of
any therapy you are providing to the patient. So, if you are still in the empirical therapy
period and you see that your patient is not having any positive reaction to the antibiotic
therapy you are giving to them, reevaluating the empirical formula is mandatory. But you
should wait at least 8-12 hours to see if the empirical therapy works or not.

Germ/Disease
With Germ Theory, we used to link one germ to one disease. Anthrax caused by
Bacillus anthracis; Tuberculosis caused by Mycobacterium tuberculosis etc. (one by one).
Whereas today we know that one disease could be caused by more than one
microorganism. For example, meningitis could be caused by N. meningitidis, S. pneumoniae,
Enteroviruses, TosV. So, when we have the clinical suspicion of a disease, we know that we
might have to deal with more than one germ.

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We have 2 different possibilities to set up the workflow in the clinical microbiology laboratory.

1. Classical Microbiology
Cons
 Growth is an issue.
-Sample transport and conservation
-Media
-Antibiotic therapy: Can influence the results of your workflow.
-TAT (Turnaround time) is longer than 24 hours as bacteria need to grow.
-Minimal germ load is required otherwise you will not get any results.
 You can detect only monomicrobial infection.

Pros
 AST (Antimicrobial susceptibility testing) can provide MIC (minimum
inhibitory concentration). That means it provides the number that
corresponds to the concentration of a drug that is capable of stopping the
growth of germ in vitro.
 You can detect living (pathogenic) germs

2. Molecular microbiology
Pros
 Growth is not an issue
-It has a very short TAT (less than 1 hour)
-You can detect germs with very low LOD (limit of detection)
-It is possible to detect multiplexing that means polymicrobial infections
Cons
 Inhibition of PCR
 You need an internal control that can state that your reaction works properly.
 You need to have control of the efficacy of the NA (nucleic acid) extraction
process.
 You have a limited pattern of genes in your reaction. That means you can
just identify a limited set of pathogens or a limited set of genes potentially
related to antimicrobial resistance.
 Another very weak point is that you detect only portions of DNA/RNA. You
are not detecting germs. Who cares about antibiotics! You can treat the
patient with a lot of antibiotics and the genes are still there. So ongoing
therapy does not at all influence molecular microbiology.

Can you just take for granted the detection of 16S RNA genes of Staphylococcus
aureus for your patient suffering from Staphylococcus aureus infection? The answer is NO!
You need to interpret these results in the light of the clinical picture of your patient in a very
critical way. When the professor started working with molecular microbiology technologies
roughly 20 years ago, they were very happy because they were able to work with the
primary blood sample for sepsis and they were so excited to detect Staphylococcus aureus
in a patient who underwent haematological stem cell transplantation. As anything you find in
these patients’ blood is potentially very dangerous, they treated the patient for
Staphylococcus aureus infection, but they did a mistake by doing so without knowing.
Haematologists sent another blood sample after 12 days and RNA was still there, but the
patient was completely afebrile and healthy. The clinical microbiology lab of the professor
said the patient is still infected but the haematologists opposed this as there were no clinical
signs of infection. Nowadays this is not a problem because we know DNAemia exists.
When you detect living bacteria in patients’ blood you can say the patient has bacteremia. If
you work with molecular biology, you can detect portions of DNA in a patient’s blood, and

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this is called DNAemia, which is completely different from bacteriemia from the clinical point
of view.

 With molecular microbiology, we are detecting genes. In general, very

complex picture of genes deriving from the microbiome, virome and so many
…omes that we have in our body. So, you need to pick up the pathogenic
genes to be effective.

The Importance of Molecular Microbiology

Molecular Microbiology is based on the concept of the genome of every single
bacterium has a region called the 16S rRNA gene. This portion of the genome has unique
sequences of DNA. Each one of these sequences can be used to identify the bacteria. We
target our PCR amplification on the 16S rRNA genes so we can identify specifically all the
bacteria we are dealing with. We have thousands of sequences we can find in databases so
we can just construct the reaction according to the most represented sequences of the 16S
rRNA gene. The secret of this technology is that you need to target this unique gene. Each
bacterial species has this kind of signature that can be distinguished from another.

We have plenty of different technologies and 3 of them represented during the class are:

1) With Film Array Blood Culture Identification (BioFire) Panel, we get a mixture of
Gram+, Gram-, fungi, and some antibiotic resistance genes (mec A, van A/B and
KPC).

2) By using Unyvero P55(Curetis AG) you get more or less the same panel of bacteria,
but you will get a lot more antimicrobial resistance genes.

3) You can use GeneXpert Carba-R if you don’t care about the identification of the
bacteria. The sample is taken by rectal swabs, and you get the results in only 48
minutes. You will identify whether the bacteria you are dealing with has one of five
classes of carbapenem resistance genes. Sometimes under the clinical point of view,
this approach is more than enough for a fast response and start of the treatment as
soon as possible. Sometimes we only care about whether the bacteria we are
dealing with have carbapenem resistance genes (e.g. blaNDM, blaKPC etc) so we
know if we can use carbapenem in this case. If one of these genes is present you
cannot use carbapenem. Of course, at the same time, the microbiology lab is
classically performing the antibiotic susceptibility test, but bear in mind that you are
dealing with a suspected sepsis patient, so you cannot wait for the antibiotic
susceptibility results as your patient has 12 hours to get the correct treatment.

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All the technologies we mentioned above work by combining different panels.

On the graph above, on the x-axis, we can see the most common 20 different
species of bacteria isolated from the septic patients in Europe and you see the
percentage of patients who have these bacteria on top of each column. So for
example, if you work with a technology that is only capable to pick up E. coli, you will
cover 28.6% of your aetiology. Whereas if you work with a technology that can cover
the 6 most common bacteria you will cover 68% of sepsis cases. If you have a
technology that can pick up all the most common 20 different species of bacteria, you
will cover 94% of sepsis cases.

How the syndromic panels should be constructed?

 Syndromic panels should evolve to include additional pathogen targets. This
is very difficult because all these technologies need to have CE-IVD (in vitro
diagnostics) mark meaning that these technologies have been approved by
the International Regulatory Agency and that they can provide useful in vitro
diagnostic information. Technologies with no CE-IVD mark would cause
laboratories to lose their certifications and get into trouble in law. When you
want to modify a panel, you need to go again under the CE-IVD marking
procedure which takes months.

 Syndromic panels should be flexible in design to better meet the user’s

needs. For example, if I can get the results I need, by covering the first 10
most common bacteria species which covers 83%, I can save a lot of money
by not including the rest of the 10 species. It is also important to be able to
customize the panel. Meaning that according to the epidemiological data I
have, the most realistic etiology could be Morganella morganii so I should
check for that. An example could be if you have a patient that has been
exposed to the dairy product which was purchased from a farmer who later
has been found out that had Campylobacter jejuni and has a fever, of
course, you should check for Campylobacter jejuni in the lab as it is the most
likely etiology for your patient.

 Syndromic panels should have shorter run times, less complexity, and be
placed near the point of care.

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 Syndromic panels should be expanded to include more antimicrobial

resistance determinants and phenotypic susceptibility results.

Emerging Technologies for Molecular Diagnosis of Sepsis

They are used in diagnosis from primary whole blood meaning that we are excluding
the use of blood cultures. In the table, we can see different technologies. Some of these are
still in the market but none of them have found a wide application in the workflow of the
laboratories. The detection limit of these technologies is basically based on PCR. The
detection limit is extremely low (they are extremely sensitive).
We can see 4 different technologies (IRIDICA, SeptiFast, SeptiTest and U-dHRM).
Let’s take IRIDICA as an example. It takes 6 hours, and you can have 6 samples per run.
The best possible one for several samples per run is SeptiTest which is 12, but this is still
very low, as the professor’s laboratory receives 100 blood cultures per day for example. This
is not feasible.

In the picture above we can see a timeline of sepsis technologies and where they fall
compared to the gold standard of blood culture and the 1–3-hour critical time for affecting
clinical decision making. None of these sophisticated technologies can have an impact in the
first 3 hours as their turnaround times are longer than 3 hours. SeptiCyte and U-dHRM may
be further optimized to provide results in a shorter time frame.

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This paper was

released 3 years ago
which evaluated one of
the most promising
technologies at the
time called IRIDICA.
The Iridica technology
was based on a very
wide panel of
detectable pathogens
(more than 800
different species). The
problems were that it
didn’t have more than 8
samples per run and
lasted 8 hours. So, you
had to select the right
patient to use this
technology. The other
problem of IRIDICA
was that its sensitivity
was extremely high. Its
detection limit was
0.25-128 CFU/ml (it
could detect 1 bacteria
in 1 ml). (You can
check slide 21 for more
detail)

If we look at the graph on the left, we can see several bacteria identified by IRIDICA in
grey and several bacteria identified in black by standard blood culture.

In the part marked by red parenthesis, we can see bacteria that are only identified by
IRIDICA. A possible explanation could be that these bacteria are not able to grow in blood
culture. But it is not the case as we know that Streptococcus or Yersinia species can grow in
blood culture. Also, very few of these patients are treated with antibiotics, so the presence of
ongoing antibiotic therapy is not the right explanation either.

If we look at the part marked by green parenthesis, we see bacteria that could only be
identified by blood culture and not by IRIDICA. As they can grow in blood culture, they
should have enough bacterial load to be picked up by PCR (IRIDICA). There was no clear
explanation for this.

They also identified very strange, uncommon bacteria that even the professor hasn’t
heard before. They later found out that these strange bacteria were frequently found in
rubber trees. This technology was too sensitive that it identified these strange bacteria in the
rubber cap which contained a little bit of DNA that was originally in the rubber tree.
Due to these reasons soon after the publication of this paper the company stopped the
production of IRIDICA. But the professor thinks that in a very short time we will be able to
move in the direction of working with this kind of technology in our routine work because that
is the only way to save more patients from the very severe prognosis.

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