SEMINAR 2 - Molecular Diagnosis of Fungal Infections

- Asthma

- HIV/AIDS

- Cancer

- Organ transplantation

- Corticosteroid Therapies

Serious fungal infections often occur as complications of these:

COPD

What has the highest incidence of life-threatening invasive Aspergillosis?

ICU patients, Lung Cancer, Hematologic Malignancies

After COPD, what are the succeeding high incidence cases of invasive Aspergillosis?

Invasive Aspergillosis and Invasive Candidiasis (Candidemia)

What are the top 2 in rankings for crude mortality of severe fungal disease?

Low sensitivity, Time-consuming, Contamination risk

Factors that can delay treatment and may lead to higher hospital costs

Direct Microscopy

Identifying specific morphological patterns help differentiate between various histopathological diagnoses of invasive fungal infections. However, this alone does not provide specific identification

Blood Cultures

Low sensitivity for diagnosing Candidemia

True

T or F: Non-blood cultures are non-specific

False

T or F: For Aspergillosis, cultures from respiratory tract secretions are very sensitive

a. Sputum

b. Bile/bronchoalveolar lavage

Aspergillosis is recovered from:

a) only 8-34% of patients with invasive Aspergillosis

b) 45-62% of such patients

Immunocompetent

Recovery of Aspergillus from respiratory tract often indicates colonization in ____ patients

Immunocompromised

Recovery of Aspergillus from respiratory tract strongly suggests invasive disease in ____ patients

Molecular Methods

These methods improve accuracy and quickness of fungal infections diagnosis

Taq Polymerase

Components of PCR: Enzyme that builds new strands

dNTPs (deoxyribonucleoside triphosphates)

Components of PCR:

- Raw materials used by the enzyme to synthesize new DNA strands

- Arranged based on the template strands as a guide

Primers

Components of PCR:

- Short custom-made sequences that adhere to the start of the target region

- Tells Taq Polymerase exactly where to start building

Buffers

Components of PCR: Help to maintain optimal condition for the reaction

Cofactors

Components of PCR:

- Help the primer adhere to the correct site

- Assist Taq Polymerase in functioning properly

- Added in the form of MgCl2

Denaturation at 95 degrees

First step in PCR where DNA is heated at a certain temperature to break the hydrogen bonds, creating two template strands for copying

Annealing at 55 degrees

- Second step in PCR where primers bracket the sequence of interest on the template in preparation for copying

- Magnesium helps these primers with its positive charge which stops the DNA and primers from repelling, while the buffer stabilizes the hydrogen bonds

Extension at 72 degrees

The last step of PCR where Taq polymerase moves along the template strand starting from the primer site, in the 5' to 3' direction, adding dNTPs to synthesize a new complementary DNA strand

Gel Electrophoresis

This is used after PCR commonly for genetic testing and infectious disease diagnosis

Real-Time/Quantitative PCR (RTQ-PCR)

This test uses reverse transcriptase to build DNA from RNA templates

Primers, Fluorescent Probes, DNA Polymerase

Components of RTQ-PCR

Fluoresence Detection

What is the additional step in RTQ-PCR where a signal is emitted as DNA is amplified

SYBR Green

This fluorescent reagent only attaches to dsDNA

TaqMan Probe

This fluorescent reagent is a short sequence that attaches to a specific target sequence that contains a glowing reporter component and a quencher

Ct Value

In an amplification, this is the point where the amplified product exceeds the threshold

True

T or F: Ct value and DNA concentration have an INVERSE relationship (low Ct value, high DNA conc)

Nested PCR

This modification of PCR is designed to improve specificity and sensitivity with 2 primer sets and 2 successive PCR reactions

- Primers

- Taq polymerase

- dNTPs

- Buffer

- Magnesium chloride

Master Mix components

Specific and Non-Specific

The first set of primers (outer primers) in nested PCR are used to shorten DNA containing the target region. This reproduces both ____ regions

Specific

The second round in nested PCR uses the products from the first amplification, where inner primers bind only to the ____ region, eliminating the other segments

Loop-Mediated Isothermal Amplification

- This rapid DNA amplification technique is isothermal at 67 degrees C

- It uses 4 to 6 primers to amplify DNA

FIP, F3, BIP, B3, Loop Forward, Loop Backward

What are the 6 primers used in LAMP, starting with the essential primers in the order they are used

Sputum

This is the sample used in LAMP for pathogen detection

DNA Polymerase

LAMP uses this enzyme with strand displacement for synthesis of new stands

F2c; F1c

In strand invasion, FIP binds to the ___ region to initiate synthesis (extension). While the ___ remains unpaired forming a 5' overhang

F3c

In step 2 of LAMP, F3 binds to the ___ upstream of F2, displacing the previous strand containing FIP to form a loop at one end

Self-Hybridization

The loop that forms in LAMP is due to this reaction between F1 and F1c

B2c; B1c

In reverse side initiation, BIP binds to the ___ region to initiate reverse synthesis causing the ___ portion to form a loop by hybridizing with B1

Dumbbell-shaped DNA Structure

What is the result of step 3 in LAMP?

Concatemeric DNA Products

The last step of LAMP uses the dumbbell DNA as a template, where loop primers speed up DNA synthesis resulting in multiple ____

1. ITS2

2. Clinical

3. Colorimetry, fluorescence, gel electrophoresis

Fallahi et al. (2019, Iran)

Target gene:

Specimen:

Detection:

1. anxC4

2. Sputum

3. Colorimetry, LFB, turbidity

Jiang et al. (2021, China)

Target gene:

Specimen:

Detection:

1. 5.8S rRNA, ITS1, ITS2

2. Blood

3. Fluorescence

Lim et al. (2022, Korea)

Target gene:

Specimen:

Detection:

Aspergillus; ITS1/ITS2/5.8S rRNA

anxC gene is commonly used for ___ while ____ are for Candida

Sanger Sequencing

A method used for determining the nucleotide sequence of DNA with a mechanism that uses a single primer to initiate DNA synthesis

Chain Termination Method or Dideoxynucleotides Chain Termination Sequencing

Other names for Sanger Sequencing

Frederick Sanger and colleagues, 1977

Who developed Sanger sequencing, and what year was it developed?

1. DNA sequence for chain termination PCR

2. Size separation by gel electrophoresis

3. Gel analysis and determination of DNA sequence

State the 3 steps of Sanger Sequencing

Chain Termination PCR

What special type of PCR is used in the first step of Saanger Sequencing that involves the addition of modified nucleotides called dideoxynucleotides (ddNTPs)?

True

T or F: In Sanger Sequencing, a low ratio of ddNTPs to normal dNTPs is used.

Millions to billions of DNA fragments terminated at random lengths by ddNTPs

What is the result of the first step of Sanger Sequencing?

Manual Sanger Sequencing

Determine if this is MANUAL or AUTOMATED Sanger Sequencing:

4 separate PCR reactions, each with only one type of ddNTP:

- ddTTP

- ddATP

- ddGTP

- ddCTP

Automated Sanger Sequencing

Determine if this is MANUAL or AUTOMATED Sanger Sequencing:

All ddNTPs are mixed in one reaction, and each ddNTP is tagged with a unique fluorescent label

Size

In the second step of Sanger sequencing, chain-terminated fragments are separated by what characteristic through gel electrophoresis?

Positive

In gel electrophoresis, DNA is negatively charged and moves toward the ______ electrode

Fragments are arranged from smallest to largest, bottom to top

What is the final results in the second step of Sanger Sequencing?

True

T or F: Smaller fragments move faster, while larger fragments move slower.

Manual Sanger Sequencing

Determine if this is MANUAL or AUTOMATED Sanger Sequencing:

Products from the 4 PCR reactions are run in four separate lanes with each lane corresponding to a specific ddNTP

Automated Sanger Sequencing

Determine if this is MANUAL or AUTOMATED Sanger Sequencing:

All fragments run in a single capillary gel inside the sequencing machine

bottom to top

In the third step of Sanger sequencing, the gel is read from _______ with each band corresponding to a specific nucleotide

5' to 3'

The synthesis in Sanger Sequencing is 5' to 3', so reading the band gives the _____ sequence

Manual Sanger Sequencing

Determine if this is MANUAL or AUTOMATED Sanger Sequencing:

User reads all four lanes at once

Automated Sanger Sequencing

Determine if this is MANUAL or AUTOMATED Sanger Sequencing:

A computer reads each band using fluorescent detection via a laser

Chromatogram

This is the output of Automated Sanger Sequencing that shows fluorescent peaks for each base along the DNA sequence

Sanger Sequencing ; PCR

In ______, the goal is to generate every possible length of DNA up to the full target length, while the goal of ______ is to duplicate the entire DNA sequence

True

T or F: PCR is used to amplify DNA in its entirety, and fragments of varying lengths may appear, but only by accident.

Next-Generation Sequencing (NGS)

A high-throughput, rapid, and cost-effective method that allow simultaneous sequencing of millions of DNA fragments such as whole genomes.

Massive Parallel Sequencing

Another name for Next-Generation Sequencing

- Genomics

- Transcriptomics

- Metagenomics (for comprehensive studies)

State the 3 applications of Next-Generation Sequencing (NGS)

1. Sample Extraction

2. Library Preparation

3. DNA Sequencing

State the 3 steps of Next-Generation Sequencing

1. Amplification

2. Addition of sequencing adaptors

State the two steps involved in library preparation performed in Next-Generation Sequencing

Library

This is produced in the PCR ampificatio step in NGS that is a collection of specifically sized DNA fragments compatible with the sequencing system

Adaptor ligation step

State the step in NGS wherein amplified DNA/cDNA fragments (amplicons) are bookended with specific oligonucleotide sequences

Parallel sequencing

DNA sequencing in NGS is performed via what method using an NGS platform?

Sequencer

This is the instrument used in NGS that reads the nucleotides one by one after loading of the library

Fluorescently labeled nucleotides

These are used in NGS to synthesize a complementary strand for each fragment.

wavelength ; intensity

In NGS, the fluorescence _____ and _____ determine the sequence of the templates

Third-Generation Sequencing

This method can read extremely long reads (up to several million bases) and it sequences DNA in real-time as it passes through a nanopore or during polymerase synthesis

- Oxford Nanopore

- PacBio SMRT (Single Molecule Real-Time)

2 examples of technologies using Third-Generation Sequencing

regions ; structural

Third-Generation Sequencing is useful for resolving complex genomic _____ and studying ____ variations

PacBio SMRT Sequencing

A single molecule and real-time sequencing technology that uses a closed and circular single-stranded DNA template

No PCR required

laser ; camera

In PacBio SMRT Sequencing, a _____ activates fluorescence signals, while a _____ system records the color and duration of the emitted light in real time

Zero Mode Waveguide (ZMW)

In PacBio SMRT Sequencing, the flow cell is equipped with a?

True

T or F: In PacBio SMRT Sequencing, base incorporation time is longer due to base modifications

Inter-pulse duration

In PacBio SMRT Sequencing, this can indicate a DNA modification event

Nanopore Sequencing Technology (ONT)

This method uses a nanopore inserted in an electrically resistant membrane that is then applied with electrical potential.

disruptions

In ONT, _____ in the current t indicate the identity of a specific molecule

hairpin structure

The DNA preparation of ONT includes a ______ to ligate double strands, enabling continuous reading of both strands

Polymerase / Helicase

In ONT, this binds and guides DNA into the pore

Ultra-Long Reads (ULRs)

Product of ONT that is above 300 kilobases, some close to 1M base pairs

True

T or F: PCR has high detection rates for fungal infections

• Standardization of techniques

• Interpretation of results

• Cost-effectiveness

• Emerging fungal pathogens

Challenges in Molecular Diagnosis of Fungal Infections

Rapid, Point-of-Care Tests

Identify the Future Direction of Molecular Dx of Fungal Infection:

- Quick results

- Require minimal lab infrastructure

- Suitable for resource-limited settings

Integration into Clinical Practice

Identify the Future Direction of Molecular Dx of Fungal Infection:

- Enables early diagnosis

- Supports personalized medicine

- Assists in surveillance and outbreak investigations

Artificial Intelligence and Machine Learning

Identify the Future Direction of Molecular Dx of Fungal Infection:

- Can assist in data analysis, predictive modeling, and image analysis

- Seen as the future of molecular diagnosis

- LAM (Lipoarabinomannan assays)

- Lateral Flow Assays

2 examples of Future Rapid, Point-of-Care Tests in Molecular Dx of Fungal Infections

Tang et al. (2016)

Spx: Bronchoalveolar lavage, blood

Target: anxC4

Identify the Specimen and Target gene in the study of Tang et al. (2016)