Professional Practice in Biomedical Science: Medical Microbiology and Virology Lab

Learning Outcomes and Introduction

• Presenter: Mark McGimpsey, Biomedical Scientist, Microbiology Laboratory, Royal Group Hospitals.

• Core Objectives:

  • Describe procedures and practices used in a virology lab for detecting and monitoring infection.

  • Compare and contrast the mechanisms of action for various diagnostic tests.

  • Explain the advantages and disadvantages associated with different testing methodologies.

  • Critically appraise the necessity for the selection and verification of tests.

Viral Disease Mechanisms

• Cellular Entry and Replication: Viruses must enter host cells and exploit host cell functions to replicate their viral nucleic acids.

• Cytopathic Effect (CPE): This refers to the morphological changes and host cell death resulting from viral growth within the cells.

• Symptomatology: Symptoms often arise from the immune response triggered by the virus-induced CPE.

• Oncogenesis: Viruses can cause a loss of control regarding host cell replication, which may result in the formation of tumors.

Specimen Types and Diagnostic Requirements

• Respiratory Infections: Requires nasal swabs, throat swabs, or sputum.

• Gastrointestinal (GI) Infections: Requires faeces specimens.

• Vesicular Rashes: Requires vesicle swabs.

• Hepatitis and AIDS: Requires clotted blood samples.

• Central Nervous System (CNS) Infections: Requires Cerebrospinal Fluid (CSF), throat swabs, or faeces.

Overview of Diagnostic Methods

• Electron Microscopy: Rarely used in current practice.

• Histopathology: Slides are examined by a Histopathologist for characteristic cellular appearances.

• Virus Isolation (Culture): Historically involved cultivation in hens’ eggs, animals, or cell lines; however, this is no longer used in standard virus laboratories.

• Serology: Currently the mainstay of viral diagnosis.

• Molecular Methods: Increasingly widely used alongside serology.

Principles of Serological Testing

• Definition: Serology is the detection of antibodies produced by the immune system in response to an infection.

• Mainstay Diagnosis: It is the primary method for viral diagnosis but can also be used to detect specific antigens, such as the Hepatitis B surface antigen ($\text{HBsAg}$).

• Immunoglobulin Dynamics:

  • Immunoglobulin M (IgM): Produced first and is short-lived. Its detection serves as an indicator of an acute infection.

  • Immunoglobulin G (IgG): Levels rise later but persist for longer periods. Detection of IgG can be sufficient for a definitive diagnosis in certain cases, such as HIV.

• Statistics: In the year 2019-20, the virus lab conducted $257,000$ tests. This volume exceeded molecular testing at the time, although molecular testing increased significantly in 2020/21 due to $\text{SARS-CoV-2}$.

Specific Serological Techniques

• Agglutination Methods:

  • Haemagglutination and Particle Agglutination: Used for tests like $\text{TPHA}$ and $\text{TPPA}$.

  • Mechanism: A microtitre plate is used where Red Blood Cells ($\text{RBC}$) or particles are sensitized (coated) with an antigen (e.g., $T. pallidum$). This is mixed with diluted patient serum.

  • Positive Result: If antibodies are present, they agglutinate the particles, which spread out and cover the bottom of the well uniformly.

  • Negative Result: Particles concentrate in a tight, visible "button" at the bottom of the well.

• Complement Fixation (CF):

  • Procedure: Patient serum is exposed to a specific antigen and a complement.

  • Mechanism: If antibodies are present, the antigen/antibody complex "fixes" (removes) the complement. Consequently, when indicator red blood cells are added, lysis will not occur.

  • Positive Reaction: Red cells are not lysed and settle into a pellet.

  • Negative Reaction: Complement remains free to lyse the red cells.

  • Wasserman Reaction: A diagnostic CF test used historically to detect antibodies to the syphilis-causing organism $Treponema$.

• Enzyme Immunoassay (ELISA):

  • Enzyme-Linked Immunosorbent Assay: Features antigens or antibodies immobilized on a microtitre plate.

  • Indirect ELISA: Antibody of interest binds to the plate; after washing, a second anti-human antibody (bound with an enzyme) is added. A substrate is introduced and hydrolyzed by the enzyme, producing a color change (e.g., yellow wells).

• Immuno-chromogenic Assay (Lateral Flow Test):

  • Example: Urinary antigens for $Strep. pneumoniae$.

  • Mechanism: Sample is drawn by capillary action; antigens bind to antibodies attached to colored markers. This complex binds to a specific antibody fixed at the test line, concentrating the color. A control line ensures the sample passed through correctly.

• Immunofluorescence:

  • Fluorescent Treponemal Antibody (FTA): Specifically used for $Treponema pallidum$.

  • Mechanism: A slide coated with the organism is treated with patient serum. Matching antibodies bind and are visualized using a fluorescein-labeled anti-immunoglobulin under an ultra-violet ($\text{UV}$) light microscope.

• Electrochemiluminescence (ECLIA):

  • Mechanism: A quantitative method measuring antigen or antibody based on changes in the $ECL$ signal before and after an immunoreaction.

  • Equipment: Utilized by modern analysers such as the Roche Cobas e602.

Infections Detected via Serology

• Syphilis

• HIV

• Hepatitis A, B, C, and E

• Rubella

• Measles

• Varicella Zoster Virus ($\text{VZV}$, causing chickenpox and shingles).

• Current Trends: Many $IgM$ detection methods are being replaced by $\text{PCR}$ for acute diagnosis (e.g., $\text{VZV IgM}$ was discontinued several years ago).

Molecular Diagnostics: Polymerase Chain Reaction (PCR)

• Status: Established as the "gold standard" in virology and increasingly in microbiology.

• History: Developed in 1983.

• Mechanism: Detects the nucleic acid of a pathogen, regardless of whether the pathogen is alive or dead.

• Attributes:

  • Sensitive: Amplifies tiny amounts of target nucleic acid to detectable levels.

  • Specific: Detects only the presence or absence of the specific target pathogen.

• Amplification Mathematics:

  • Cycle 1: $2^1 = 2$ copies.

  • Cycle 2: $2^2 = 4$ copies.

  • Cycle 3: $2^3 = 8$ copies.

  • Cycle 30: $2^{30} \approx 10^9$ copies.

SARS-CoV-2 PCR Methodology

• Sample Types: Throat/nasal swabs or sputum.

• Biohazard Classification: Provisionally designated as a Hazard Group 3 ($\text{HG3}$) pathogen; all samples must be processed in a safety cabinet.

• Process:

  1. Lysis: Lysis buffer is added to release cellular nucleic acids.

  2. Reverse Transcription: Since $\text{SARS-CoV-2}$ is an RNA virus, reverse transcriptase converts RNA into complementary DNA ($\text{cDNA}$).

  3. Denaturation: $cDNA$ is heated to $90 - 95^\circ\text{C}$ to separate strands.

  4. Annealing: Primers bind to target sequences (envelope, nucleocapsid, or spike protein) at $55^\circ\text{C}$.

  5. Extension: Taq polymerase enzyme extends the strand at $75^\circ\text{C}$.

• Real-Time PCR: Uses a probe with a fluorescent dye and a quencher. Taq polymerase cleaves the probe during extension, releasing the dye from the quencher and emitting a detectable signal.

Data Interpretation and Lab Logistics

• Regional Virus Laboratory (RVL) Statistics:

  • February 2021: $3,626$ tests per week.

  • January 2022: Approximately $3,000$ tests per day.

• Amplification Curves:

  • Sigmoidal Curve: Indicates a positive result.

  • Cycle Threshold (CT) Value: The point where the signal rises.

    • Low CT: High viral load.

    • High CT (>35): Questionable; a repeat sample is often requested.

    • Flat Line: Negative result.

Comparison of COVID-19 Testing Technologies

Evaluation of the Lumira Dx Pilot

• Analytical Performance: $836$ patients tested; $301$ compared against lab-based PCR.

• Discrepancies: When PCR (\text{CT} < 40) was the comparator, Lumira Dx had 7 false negatives and 17 false positives.

• Diagnostic Metrics (Adjusted):

  • Sensitivity: $85.71 \%$ (Note: 3 false negatives were early-stage and asymptomatic).

  • Specificity: $93.93 \%$.

  • Negative Predictive Value (NPV): High ($99.21 \%$ to $99.85 \%$ depending on prevalence).

• Chief Medical Officer Guidance (Dr. Michael McBride): Lumira DX is a "rule out" test for rapid identification of negative patients. It detects the nucleocapsid protein antigen as a measure of infectiousiveness. Positive results must be confirmed by a second alternative PCR test.

PCR Sensitivity vs. Public Health Utility

• Sensitivity Concerns: PCR can detect degraded viral signatures (RNA fragments from sub-genomic genes like E and N) for weeks after a person is no longer infectious.

• Public Health Utility: Low-sensitivity tests (like Lateral Flow or LAMP) may be better for identifying current infectiousness by only detecting higher viral loads.

• Clinical Utility: $\text{RT-qPCR}$ is preferred for identifying both active and historical infections within the last few weeks.

Case Study: Microbiology Discrepancy (September 2024)

• Sample: Enteric sample.

• Initial Findings:

  • PCR: Positive for $Shigella$, not detected for $Salmonella$.

  • Culture: $Shigella$ not isolated; $Salmonella enterica$ spp isolated from enrichment only.

• Investigation: Repeat PCR and culture confirmed initial results. Selenite neat culture was negative for $Salmonella$ contamination. Serosep confirmed $Shigella$ amplification was valid.

• Final Reporting:

  • Shigella: Reported as "PCR positive" but "culture not isolated." Note: Consider false positive or low numbers/non-viable organism.

  • Salmonella: Reported as "PCR not detected" but "culture isolated from enrichment." Note: $Salmonella$ was present but below the PCR detection limit.