University of Essex
School of Biological Science
Discuss how an understanding of symptoms of
microbial diseases helps to design diagnostic
procedures in Medical Microbiology laboratories
Essay in Advanced Medical Microbiology (BS318), Msc Molecular
Medicine
By: Priyankaa Goordyal
1
Introduction
The aim of this essay is to provide an understanding of the role of
medical microbiology in the diagnosis of a patient suffering from an
infection. Here, we describe the patient pathway from when the patient
presents his symptoms to when the patient receive treatments.
Additionally, we review 2 case studies where the communication
between clinicians and Biomedical Scientists helped in the diagnosis
and treatment of microbial infections. Furthermore, we explored on how
biomedical scientists are trained and how new molecular tools are
assessed before it is implemented in a medical laboratory.
Discussion
The incubation period of a microorganism is a term used to define the
time from when a patient is exposed to a microorganism to the time a
patient start displaying symptoms of an infection (Nishiura, 2007). In
order to make a differential diagnosis and provide effective treatments to
a patient suspected of having an infection, the clinician reviews the
patient’s history and carries out a physical examination of the patient.
Information such as age, height, weight, BMI (Body Mass index),
previous medications and current undergoing illness are recorded. In
2
addition other key and relevant clinical details are recorded to aid
diagnosis. For example if the patient is suspected of contracting sexually
transmitted diseases sexual history is also recorded. If a patient is
suspected of having Food Borne infection, food consumption is
recorded. Additionally, if the patient has recently been travelling and is
suspected to have contracted an infection abroad, this is also recorded
on the clinical details to aid diagnosis (Herrett et al., 2010). In order to
confirm the diagnosis of a disease or determine the causative agent of
the disease as well as choose appropriate treatments, clinicians have to
send samples to a Medical laboratory. The clinicians collect appropriate
specimens according to the signs and symptoms of patients. The
clinician has to ensure that they follow guidelines from the laboratory to
effectively take samples at the appropriate site of infection. For example,
using the appropriate technique to collect sample, avoiding cross
contamination and making sure that there is enough sample for the
laboratory to process. If such criteria are not met, diagnosis is difficult
and such samples may be rejected by the laboratory(Washington, 1996).
When a clinician submits a sample and requests for investigations to be
carried out by a Medical laboratory, the clinical details, type of specimen,
date and time of collection is recorded in the request form. If a virulent
microorganism is suspected, the clinician has to indicate this on the form
as the Biomedical Scientist would have to treat the sample differently
3
and work under a safety cabinet. The clinical details on the form help the
laboratory to determine the appropriate techniques (e.g culture) to
isolate the microorganism. The primary role of a medical microbiology
laboratory is to aid clinicians to identify the causative agent of the
infection and help determine appropriate antimicrobial susceptibility
profile to help clinician treat or modify the antimicrobial therapy of
patient. By providing appropriate antimicrobial profiles, the microbiology
laboratory aims to reduce the rate of antibiotic resistance. Additionally,
the microbiology department plays an important role for infection control
in hospital by detecting nosocomial infection (hospital acquired infection)
(Kolmos, 2001) and epidemiological studies for surveillance of
microorganisms and to prevent the emergence of outbreaks. The
microbiology department usually reports any new unusual occurrences
to Public Health England to monitor the pattern of infectious diseases
(Duerden, 1994).
The microbiology laboratories in the UK receive several types of
specimens depending on the type of infections and type of transmission.
For example for Upper respiratory tract infections occurs from mostly
airborne transmission. The types of samples include throat swabs, nose
swabs, ear swabs, eye swabs, mouth swabs and pre-nasal swabs.
Specimens for Lower respiratory tract infections include bronchial
4
washings, pleural effusions, sputum, urine, tracheal, trans tracheal and
bronchial aspirates and this is could be transmitted via hospital-acquired
infection. For food borne or gastrointestinal tract infections, the mode of
transmission is mostly via contaminated food. Samples include stool
samples. Finally, the common specimens for sexually transmitted
diseases include mouth swabs (Oral sex), eye swabs from neonates (e.g
Sexually transmitted disease transmitted during pregnancy), vaginal,
endocervical and ureteral swabs and fluid samples (from sores and
vaginal discharge) (Great Ormond Street Hospital, 2016).
Swabs are usually stored and transported in amine medium with
charcoal. This transport medium contains antitoxins so bacteria will not
multiply or kill themselves. Other samples can be stored in the fridge so
that bacteria cannot multiply. Biomedical Scientist has the knowledge on
how to deal with the sample promptly and efficiently (Rosa-Fraile et al.,
2005). Fluids swabs are preferred as bacteria can grow better in fluids
than swab and therefore isolation is easier. The request forms of the
sample each have a bar code and specimen received from different sites
each have different standardize tests for the isolation of specific
bacteria. The tests are pre-determined by the sample site, clinical details
of patients and knowledge of the Biomedical Scientists. These tests are
standards for each infection and are based on evidence-based practice,
5
epidemiological statistics, and knowledge of clinicians and experienced
Biomedical Scientists. Evidence based practice is when the laboratory
employs a new test/method based on research to improve work-load
and improve patient outcome (Giocoli et al., 2009; McKibbon, 1998).
Additionally, for some patients (for example Immunocompromised
patients, diabetics and pregnant patients), further tests are added to
standardize test, as the immune system is lower on these patients and
are therefore more susceptible to other types of infections. This is also
based on evidence-based practice and is done to ensure better outcome
for the patient. For example, sputum samples are analyzed for
microorganisms
pneumoniae,
such
as Haemophilus
Staphylococcus
aureus,
influenzae,
Moraxella
Streptococcus
catarrhalis
and
Legionella in patients suffering from respiratory tract infections (such as
bronchitis, chest infection, chronic obstructive airways disease or
pneumonia). However, if the request form indicates the patient is
immunocompromised, further investigations and the Biomedical Scientist
needs to look for Enterobacteriaceae, pseudomonads and fungi.
Furthermore, the appearance of the sample needs to be considered. For
example, if Biomedical Scientist observes that the sputum sample is
bloody, he needs to look for Mycobacterium tuberculosis. Additionally,
when a Biomedical Scientist looks at the sample type and specimen, he
6
should know the conditions (aerobic or anaerobic conditions) and the
type of media in which the microorganism should grow. For example
microorganism from an ear swab in otitis media should be grown in
anaerobic conditions and needs to carry out a gram stain to determine if
the microorganism is gram negative or gram positive. Therefore, in order
to correctly isolate and identify microorganism, the Biomedical Scientist
needs to be skilled and experienced to make this decision. These are
examples on how the experienced and knowledgeable Biomedical
Scientist needs to check for the clinical details of the patients as well as
sample type, sample sites and sample appearance to carry out the
investigation.
Generally, most samples received in the microbiology laboratory, needs
to go through Microscopy, Culture and Sensitivity tests. For example, in
order to efficiently identify a microorganism, the Biomedical scientist
need to have an understanding of the shape, pattern of growth of
microorganisms and in some sample identify if the bacteria is gram
negative and positive to correctly identify the organism. Sometimes, this
can be a problem as some types of microorganism share similar
structures, shape and therefore further differential tests are required.
Because of this limitation and the fact that the resolution of light
microscopy are low, rapid identification of emerging infectious agents
7
are currently being identified by electron microscopy in some areas of
the United State of America (USA). This has changed to improve
diagnosis and provide better patient outcome (Hazelton and Gelderblom,
2003).Cultures are used to isolate pathogenic microorganisms from the
normal flora. For each specific microorganism, there exists, different
types of cultures to identify specific microorganism. The agar contains
specific nutrients and is kept at specific conditions for bacteria to grown.
For example, throat swabs from Bordetella species are inoculated in
charcoal agar with cephalexin and left to grow for 7 days in a moist
chamber. Again, these are the skills and knowledge that are required by
Biomedical Scientist to ensure correct diagnosis of disease.
Antibiotic susceptibility test of microorganism is important to check for
the resistance of the microorganism. Disk diffusion test are used to
assess for antibiotic susceptibility. This is where 12 paper antibiotic discs
are placed on inoculated agar surface and the agar is left to incubate for
16-24 hours at 35oC. The zone of inhibition is measured around each
antibiotic disk to the nearest millimetres. This value is then compared to
a standard interpretation chart. The results of the disk diffusion test is
qualitative and the antibiotics can be determine as Resistant,
Intermediate or susceptible (Jorgensen and Ferraro, 2009).
Additionally, E tests (gradient diffusion method) enable Biomedical
8
Scientists to report the MIC (Mean inhibitory Concentration), the lowest
concentration of antibiotic at which bacteria stop. This test enables
microbiologists to inform the clinicians of the best possible antibiotic for
the patients and informs the clinician of treatment regimes that is likely to
be ineffective regimes. Additionally, it enables microbiologists to advice
clinicians on the appropriate dose for antibiotics. In turn, the clinicians
can use pharmacodynamics and pharmacokinetics to determine the best
possible dosage of antibiotic for the patients.
Properly performed antibiotic testing can help to identify bacterial
isolates that are resistant to antimicrobial agents. Some studies
identified cases where Antibiotic Susceptibility testing was poorly
performed by technical staffs. Additionally, some studies identified
problems with technical staffs with interpreting invitro antibiotic
susceptibility test. This affected the therapeutic outcome of the patients.
Some studies also confirmed that there were no correlation between the
number of AST performed and therapeutic outcomes (Peterson and
Shanholtzer, 1992). In order to tackle this problem, USA implemented
several educational programs to improve AST policies and practices.
These
educational
programmes
consisted
of
regional
technical
workshops, National Laboratory training Network teleconferences, use of
the center for Diseases and prevention (CDC) CD-ROM on AST and the
9
CDC
Multilevel
Antimicrobial
susceptibility
testing
website.
The
implementation of these programme helped to improve the therapeutic
outcome of the patients (Counts et al., 2007).
These incidences highlight the importance of educating and training staff
properly and highlight the importance of having CPD (Continuing
Professional Development) in order to ensure best practice in
microbiology laboratories. In the United Kingdom, all staffs are given
appropriate training by the hospital. Additionally, the person interpreting
the results needs to be a HCPC (Health Care Professional Council)
registered Biomedical Scientist. A HCPC registered scientist is someone
who has been signed off on a number of competencies and has
undergone thorough training to interpret results. A Biomedical Scientist
has the relevant knowledge and experience on how to deal with the
sample, how to safe guard patients and how to ensure and maintain
fitness to practice (Pitt and Cunningham, 2011).
Additionally HCPC keeps a register of all registered Biomedical Scientist
to help employer identify qualified professionals and safeguard the
patient.
In the United Kingdom, in order to ensure the quality of work and quality
of tests performed (Quality control), the laboratory has an internal and
10
external quality control system. The internal quality controls are test are
interventions performed by individuals within the laboratory to ensure
that the results produced by the laboratory is reliable and accurate. For
example, in microscopy, two or more microscope slides are prepared as
part of standard protocol.
Additionally where machines are used,
positive and negative controls have to give appropriate data otherwise,
the
investigations
have
to
be
repeated.
Medical
microbiology
laboratories accredited by CPA (Clinical Pathology Accreditation), have
to make sure that all pre-analytical, analytical, post-analytical stages are
included in SOP (Standard operating procedures) and that all these
stages are standardize to maintain consistency and ensure that the
results produced by the laboratory is of quality. These laboratories have
to follow international standards (ISO) set by CPA. CPA ensures that the
laboratory is competent, ensures quality of the work produced by the
laboratories through international standards as well as assesses the
laboratories through inspection. CPA as now become part of UKAS
(United Kingdom Accreditation Service), this is a strategy employed by
the government to modernise clinical pathology (Arora, 2004).
Additionally, laboratories have to participate in external quality
assurance called NEQAS (National External Quality Assurance
Scheme). The investigations performed by the laboratories are assessed
and the laboratories are given a score. The score given to specific
11
laboratories is compared to the score of laboratories. This scheme
enables laboratories to identify investigations that need improvements
and enables the laboratories to see if they are underperforming. These
tests measure accuracy of the investigations and so if the laboratory is
underperforming, they need to make improvements (Snell, 1985).
Case Studies:
Case Study 1- Ophthalmia Neonatorum
An 8 day old male infant was in outpatients for 5 days because of
purulent eye discharge on both of his eyes. The infant’s mother has a
yellowish vaginal discharge before giving birth but was otherwise
asymptomatic. The mother had sexual contact with her husband about a
month ago and denied any other sexual contact. Her husband admitted
of having a urethral discharge about the same time but has previously
been to the GP to be fully treated. The patient was provisionally
diagnosed with Neisseria gonorrhoeae. The patient was unaware of this
and remained untreated. Smears from the infant’s eye and mother’s
endocervix were sent to a microbiology laboratory. Gram staining
showed gram negative intracellular diplococci. The samples were
oxidase positive and PCR (polymer chain reaction) confirmed the
diagnosis of Neisseria gonorrhoeae (Pang et al., 1979).The neonate was
12
treated with eye drops of erythromycin (Matejcek and Goldman, 2013).
Additionally, the mother was treated with cephalosporin (Barry and
Klausner, 2009).
Case study 2- Gastrointestinal tract infection
A 24 year old man presented to a clinic with a ten hour history of
diarrhoea and a seven hour history of vomiting. On physical examination,
the patient was in mild distress, disorientated to place and time but
talkative. Additionally, the patient had sunken eyes, poor skin rigor, has
an abnormal heart rate (tachycardia) and low blood pressure (90/60).
The patient was treated with presumptive diagnosis of Cholera. The
patient was treated with oral rehydration therapy to restore electrolytes
and water level. Stool samples were sent to a microbiology laboratory.
Microscopy analysis of the sample displayed gram negative comma
shape rods. The sample was oxidase positive and was isolated in TCBS
(Thiosulfate citrate bile salts sucrose) agar. Laboratory analysis
therefore confirmed infection with Vibrio cholera (Kavic et al., 1999;
Martinez et al., 2010).
Automation
The increasing workload and pressure on medical microbiology has
caused many processes to become automated. Automation enabled
13
faster turnaround times and has increased in diagnostic value for several
reasons. For example an automated machine carries out Microscopy in
urine sample. Additionally, automatic plate spreader (BD innova) is
implemented in some laboratories to make culturing process less labour
intensive. Automation made microbiology laboratories leaner and
therefore enables for Biomedical Scientist to interpret the results when
required (Riordan et al., 2002).
Furthermore, Vitek or MALDI-TOF are automated machines which
confirms the identity of microorganism and provides the antibiotic
susceptibility profile of the microorganisms. This machine has been
implemented in many laboratories to cope with turnaround times. The
implementation of these machines has increased diagnostic value and
improved patient outcome (Ligozzi et al., 2002).
Generally, the uses of novel technologies to replace methods in
microbiology have been slow. This is because published journals that
reported novel approaches to identify microorganism was not reported
according to STARD (the standard for reporting of diagnostic accuracy)
framework. This made it difficult for laboratories to validate the methods
as specificity, sensitivity and accuracy of the novel approach was not
reported. Sensitivity is define as the smallest quantity of an organisms
14
that can be detected in the specimen and specificity is viewed as how
specific is the method for that microorganism, the highly specific a test
is, the less likely it is to cross react and more likely to be specific to a
microorganism. The introduction of new diagnostic techniques requires
robust and valid diagnostic evaluation studies for implementation
(Giocoli et al., 2009).
In many laboratories, swabs suspected of chlamydia are now analysed
by
PCR
(polymer
chain
reactions).
PCR
is
where
specific
oligonucleotides are used to detect the presence of microorganisms. In
general, chlamydia was harder to grow and culture on plates and this
affected the diagnostic value. To overcome this limitation, PCR is now
employed and therefore this method is highly specific and sensitive.
Additionally, PCR is also used to diagnose Neisseria gonorrhoeae.
Furthermore, usually plate culture techniques require 24 hours before a
diagnosis can be made. With PCR techniques, the procedure is only 20
minutes long. The techniques enable rapid diagnosis. Rapid diagnosis
ensures faster treatment and can help prevent the spread of the
infections and therefore preventing outbreaks (Rahimi et al., 2013;
Whiley et al., 2006).
15
The number cases showing antibiotic resistance have increased over
the past few years and if the new treatments are not developed, these
pathogens could spread across a population and cause outbreaks (Fair
and Tor, 2014; Ventola, 2015).
This is one of the major challenges that clinicians, microbiologist and
epidemiologist face. In order to understand antibiotic resistance and
outbreaks, the genome of bacteria needs to be analysed. Research into
this area has expanded over the last few years. Bioinformatics
databases can be used to identify the genome of a bacteria or a species
(Lin et al., 2006). Phylogenetic analysis can be used to compare the
genome of different isolates of the same species. This enables the
identification of the position at which a gene is mutated and what cause
evolution, in other words, what cause species to diverge. Understanding
divergence will enable us to identify the regions that cause bacteria to be
more pathogenic (Lin et al., 2015).
Additionally, functional structure of these virulence factors and
understanding how they work would enable the new development of
antimicrobial agents by targeting specific areas within the proteins. For
example studies have found that in food borne infection, vibrio cholera is
becoming resistant to antibiotics due its virulence factor, the presence of
a capsule in chromosome 1. Because of this people died as antibiotics
cannot enter the bacteria (Heidelberg et al., 2000).
16
With this understanding from research, novel strategies could be
employed in combination with antimicrobial agents. For example, RNAi
mediated silencing could be used to target the gene expressing the
virulence factor and sensitize resistant bacteria to the antimicrobial
agents. Additionally the presence of multiple pathogens can be detected
using resequencing DNA microarrays. These DNA microarrays can be
made based on statistics from epidemiological statistics. This method
does not require specific PCR oligonucleotide to detect pathogens. The
presence of multiple pathogens are analysed by software which
compares sequence similarity to different types of strains of pathogens.
The advantage of using this method is that it enables the identification of
coinfections and is relatively easy to interpret (Lin et al., 2006).
17
Conclusions
In conclusion, diagnosis of a microbial infection is a multidisciplinary
team effort. When a patient present with clinical symptoms, the clinician
assesses the patients and request for tests to confirm diagnosis and
determine the best possible antimicrobial treatment or modify current
treatments using the facilities of a microbiology laboratory. To process
and perform tests, a qualified Biomedical Scientist, need to assess the
appearance of the sample, the clinical details and the sample site. New
research, methodology and molecular tools are being employed in
medical microbiology laboratory to cope with demands based on
research and epidemiological studies and on evidence-based practice.
Finally, it is important to realise that clinicians and Biomedical Scientist
are not the only ones that improve the patient pathway. Researchers,
biomedical scientists, clinicians and epidemiologists, all work together to
safeguard patients, monitor pathogens, prevent outbreaks and tackle
antibiotic resistance.
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