Manchester Metropolitan University

Faculty of Science and Engineering

Department of Life Sciences
Name:
Imtnan Ahmad

MMU ID:
23733860

Submitted to:
Dr. Nasser Al – Shanti

Course:
Analytical Techniques in Biomedical Sciences

Course Code:
6ABM7134_2324_9
PCR and Serology: A Comparative Analysis of Their Diagnostic
Performance for Mycoplasma Pneumoniae Pneumonia
Pneumonia is a condition characterized by the inflammation of the lungs, which is caused by
various pathogens such as bacteria, viruses, fungi, or parasites. It spreads from person, to person
through droplets released during coughing or sneezing. The incubation period for this infection
typically lasts between 2 to 3 weeks (CDC, 2022). Among community acquired pneumonia cases
Mycoplasma Pneumoniae is a culprit especially affecting children and young adults. Symptoms
of this type of pneumonia usually appear steadily which include fever, cough, sore throat,
headache, and malaise. In some cases the infection can also lead to skin rashes, joint pain
(arthritis) heart inflammation (myocarditis) brain inflammation (encephalitis) or a type of anemia
called hemolytic anemia (CDC, 2022). Diagnosing Mycoplasma Pneumoniae pneumonia can be
challenging as culturing the organism is difficult requiring media and conditions. Therefore,
alternative diagnostic methods such, as polymerase chain reaction (PCR) and serology are
necessary.
PCR is a molecular technique that amplifies and identifies DNA sequences of Mycoplasma
Pneumoniae present in samples like respiratory secretions, blood or cerebrospinal fluid (CDC,
2022). PCR is used because of its faster turnaround time, higher sensitivity, lower risk of
contamination, and ability to detect multiple pathogens simultaneously (Figallo et al., 2017).
Moreover PCR can also be utilized to determine the susceptibility of Mycoplasma Pneumoniae
to antibiotics, which's crucial, for guiding appropriate treatment decisions (Leng et al., 2023).
Serology is an immunological technique that measures the antibody response of the host to
Mycoplasma Pneumoniae antigens, such as the P1 adhesin or the 16S rRNA (CDC, 2022).
Serology can also provide information on the immune status and epidemiology of Mycoplasma
Pneumoniae infections (Figallo et al., 2017). Serology testing can also help monitor the
effectiveness of vaccines and treatments by measuring the antibody response over time (Dos
Santos Ferreira et al., 2021).

Polymerase Chain Reaction

PCR is a technique used to amplify and detect DNA sequences of Mycoplasma Pneumoniae
from samples taken from patients, such, as sputum, throat swabs or blood. To perform PCR
oropharyngeal swabs are collected from individuals showing symptoms. DNA extraction is then
carried out using specialized kits (Hammitt et al., 2011). PCR requires a DNA polymerase
enzyme (Taq polymerase), a primer pair (complementary to the 3’ ends of the target DNA
region), and deoxyribonucleotide triphosphates (dNTPs) as the main components. PCR involves
three main steps that are repeated in cycles: denaturation, annealing, and extension.

Denaturation
In the polymerase chain reaction (PCR), the denaturation process involves the splitting of
double-stranded DNA into individual strands. This step requires the extracted DNA to be
exposed to high temperatures, usually ranging from 94-98°C. The high temperature causes the
DNA strands to unwind, creating single-stranded templates that are used for further amplification
in the PCR process (Chaudhry et al., 2013).
Annealing
The target DNA region is a specific segment of the Mycoplasma Pneumoniae genome that can
be used for identification and diagnosis. Annealing is the step where the temperature is lowered
to about 54-60°C, which allows the primers to bind to their complementary sequences on the
template DNA.
Extension
Extension is the step where the temperature is raised to about 72°C, which activates the Taq
polymerase and enables it to synthesize new DNA strands using the template DNA and the
dNTPs. Each cycle of PCR doubles the amount of the target DNA region, resulting in an
exponential amplification (Nilsson et al., 2008). The number of cycles depends on the initial
amount of the template DNA and the desired amount of the product DNA.
Detection
Typically, 25-40 cycles are performed. There are several methods available to detect and analyze
the product DNA, including gel electrophoresis, real-time PCR, and sequencing. By identifying
mutations in the 23S rRNA gene, PCR can also detect macrolide-resistant strains of Mycoplasma
Pneumoniae (Chaudhry et al., 2013).

Application of PCR
PCR offers advantages compared to diagnostic methods, like culture, serology or nucleic acid
amplification techniques (NAATs). Unlike culture, which can take weeks to provide results PCR
can deliver them within hours. PCR has the ability to detect Mycoplasma Pneumoniae DNA
when the bacteria are not viable or present in low numbers (Loens et al., 2003). Additionally,
PCR can identify strains of Mycoplasma Pneumoniae that're resistant to macrolide antibiotics by
detecting mutations in the 23S gene. The sensitivity and specificity of PCR for MP detection
vary between 80% and 100% depending on the specimen type and the specific PCR method
employed (Wolff et al., 2008). Various types of PCR methods are available for detecting
Mycoplasma Pneumoniae, including real time PCR, multiplex PCR, simultaneous amplification
and testing (SAT) and GeneXpert. Some of these methods can also detect pathogens such, as
Chlamydia pneumoniae and Legionella species within a single assay (Thurman et al., 2011).

Figure 1 Detection of Mycoplasma Pneumoniae through PCR

PCR is a powerful and widely used technique for Mycoplasma Pneumoniae detection due to its
high sensitivity and specificity. It directly targets the bacterial DNA, allowing for the
identification of even low bacterial loads. This is crucial, as Mycoplasma Pneumoniae infections
often present with low pathogen concentrations. PCR's ability to deliver rapid results, often
within a few hours, is advantageous for timely diagnosis and treatment initiation.
Limitations
PCR's usefulness is not without limitations. It requires specialized equipment, skilled personnel,
and a controlled laboratory environment, making it less accessible in resource-limited settings.
Additionally, the potential for false positives or contamination can occur during sample
processing, necessitating stringent quality control measures.
Infrastructure and Expertise Requirements
PCR demands specialized laboratory equipment, including thermal cyclers, and skilled personnel
for DNA extraction and amplification. This can limit its accessibility in resource-limited settings
where sophisticated infrastructure and trained technicians may be lacking (Tedla, 2019).
Potential for Contamination
The sensitivity of PCR makes it susceptible to contamination issues during sample processing.
Even trace amounts of DNA from external sources can lead to false positives, requiring stringent
quality control measures to ensure result accuracy (Garibyan and Avashia, 2013).
Lack of Discrimination Between Live and Dead Organisms
PCR does not distinguish between live and dead Mycoplasma Pneumoniae, potentially leading to
the detection of non-viable organisms (Soejima et al., 2008). This limitation can impact the
clinical relevance of positive results, as not all positive PCR results may indicate active
infections.
Serology Testing
Serology testing is a crucial diagnostic tool that assesses the presence and quantity of antibodies
specific to Mycoplasma Pneumoniae in the serum, utilizing blood samples collected from
patients. The antibodies include IgM, signaling a recent infection, and IgG, indicating a past or
chronic infection. Following serum separation, various methods like enzyme immunoassay
(EIA), indirect immunofluorescence assay (IFA), complement fixation test (CFT), and particle
agglutination test (PA) are employed to detect these antibodies.
EIA relies on enzyme-linked antibodies to produce a measurable color change, observed via a
spectrophotometer. IFA employs fluorescent-labeled antibodies, observable through a
microscope or fluorescence reader. CFT utilizes the complement system to induce the lysis of
antibody-coated red blood cells, with the degree of hemolysis measured by a spectrophotometer.
PA involves latex particles coated with antigens, causing observable agglutination. Test
interpretation hinges on the antibody type and level detected, with IgM indicating recent
infection and IgG pointing to a prior or chronic condition (Alibolandi et al., 2022).
Serological testing's diagnostic significance extends to confirming Mycoplasma Pneumoniae
infection, particularly through a significant rise in antibody titers between two samples taken 2-4
weeks apart (Wang et al., 2012). This comprehensive approach aids in understanding the
infection's timeline and guiding appropriate medical interventions.
Application of Serology testing
Serological testing plays a crucial role in various medical and diagnostic applications. The
serological tests detect specific antibodies or antigens to identify infections such as HIV,
hepatitis, and syphilis. Serology is vital in blood typing for transfusions, ensuring compatibility
between donors and recipients (Li and Guo, 2022). It is also employed in autoimmune disease
diagnosis by detecting antibodies attacking the body's own tissues. Serological tests are used to
evaluate vaccine efficacy and immune response. Serology also aids in monitoring the
progression of certain diseases and assessing treatment effectiveness.

Figure 2 Serological Diagnostic Testing

Serological testing measures the host's immune response to Mycoplasma Pneumoniae, providing
a complementary approach to PCR. It is generally more accessible, requiring less sophisticated
equipment and expertise. Serological tests are useful for assessing past infections and can
contribute to epidemiological studies by identifying individuals with prior exposure.

Limitations
Nevertheless, serological testing has notable limitations. It may not detect acute infections early
in the course of the disease, as antibody titers take time to develop. Cross-reactivity with
antibodies from other infections can lead to false positives or complicate result interpretation.
Additionally, variations in individual immune responses can affect the accuracy of results,
making serological testing less reliable in certain scenarios.
Delayed Detection in Acute Infections
Serological tests rely on the host's immune response, leading to a delay in the detection of
antibodies during the early stages of infection. This can result in false negatives in the initial
phase of the disease, hindering timely diagnosis and treatment initiation (Bedekar et al., 2023).
Cross-Reactivity and Specificity Issues
Serological tests may exhibit cross-reactivity with antibodies from other infections, introducing
the possibility of false positives (Papa et al., 2011). This lack of specificity can complicate result
interpretation and lead to misdiagnosis, particularly in regions with a high prevalence of other
respiratory infections.
Variability in Immune Responses
Individual variations in immune responses can affect the accuracy of serological tests. Some
individuals may produce antibodies more rapidly or at higher levels than others, leading to
inconsistencies in test results and potentially impacting diagnostic reliability (Bedekar et al.,
2023).
Critical Evaluation of the Diagnostic Techniques
The critical evaluation of the performance for PCR and Serological testing for the detection of
Mycoplasma Pneumoniae is as follows:
Criteria PCR
High sensitivity and specificity
Reliability because the DNA are detected
(Yang and Rothman, 2004).
Low and precise measurements
Imprecision providing consistent results (Yang
and Rothman, 2004).
PCR is highly accurate in
identifying Mycoplasma
Accuracy
Pneumoniae at the molecular level,
providing specificity in diagnosis
(Yang and Rothman, 2004).
Generally linear within a dynamic
range to detect increasing
Linearity concentrations of Mycoplasma
Pneumoniae DNA (Zhao et al.,
2023).
Low limit of detection that it is
Limit of Detection capable of detecting low DNA
concentrations (Zhao et al., 2023).
PCR exhibits high sensitivity,
detecting low concentrations of
Sensitivity Mycoplasma Pneumoniae DNA. It
is particularly useful in early-stage
infections (Yang and Rothman,
2004).
PCR is highly specific, targeting
unique DNA sequences of
Mycoplasma Pneumoniae,
Serological Testing
Sensitivity and specificity can
vary due to potential cross-
reactivity with other pathogens
(Haselbeck et al., 2022).
Influenced by variations in
antibody levels, affecting
precision.
Dependent on the test type; may
have false positives/negatives
due to potential cross-reactivity
and the reliance on immune
response markers (Haselbeck et
al., 2022).
Limited linearity; may not reflect
the quantity of the organism
accurately due to variations in
antibody titers.
Higher limit of detection that it
may not detect low bacterial
loads.
Serological tests, such as
ELISA, may have lower
sensitivity in the early stages but
improve as the infection
progresses, as the immune
response develops (Haselbeck et
al., 2022).
Serological tests can sometimes
lack specificity, as antibodies
may cross-react with other
 Specificity microorganisms, leading to false
positives. Specificity may be
minimizing false positives (Yang affected in areas with high
and Rothman, 2004). prevalence of atypical
pneumonia (Haselbeck et al.,
2022).
Speed Time-consuming; may take days
Rapid; results within a few hours.
to weeks for results.
Requires skilled personnel for
Skills Requirement Skilled technicians required
interpretation.
Cost Moderate to high; equipment and Moderate; reagents and
reagents. personnel costs.

Similar Diagnostics Techniques

The other Analytical techniques for the detection of Mycoplasma Pneumoniae are culturing Cold
agglutinin titer and direct immunofluorescence.
Culturing of Microorganism
Culture involves the growth of microorganisms in a controlled environment. While it is a
standard method for diagnosing infections, it can be time-consuming, requiring days to yield
results. Culture also has limitations in terms of sensitivity, as some organisms may be difficult to
grow or may not survive the transportation and storage conditions. For Mycoplasma
Pneumoniae, which has specific growth requirements, culture may not always be the most
efficient method (Daxboeck et al., 2003).
Cold agglutinin titer and direct immunofluorescence
Cold agglutinin titer and direct immunofluorescence are serological techniques that rely on the
detection of antibodies or antigens, respectively. Cold agglutinin titers are used to identify
antibodies that agglutinate red blood cells at low temperatures, a phenomenon associated with
Mycoplasma Pneumoniae infections (Shehab, 2008). However, this method may lack specificity
and sensitivity. While it can provide rapid results, it may not be as sensitive as molecular
methods like PCR.

PCR stands out for its accurate identification of Mycoplasma Pneumoniae DNA, but hurdles
such as specialized infrastructure and susceptibility to contamination underscore the need for
careful consideration in its application. Whereas, serological testing's accessibility and broader
utility provide a complementary perspective, but challenges like delayed detection in acute
infections and the potential for cross-reactivity warrant thoughtful interpretation of results. The
choice of method depends on factors such as the stage of infection, resource availability, and the
specific requirements of the diagnostic setting. Integrating multiple approaches may enhance the
overall reliability of Mycoplasma Pneumoniae detection.
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