Laboratory Diagnostics: POCTs and NAATs

Point-of-Care-Tests

• Point-of-Care Tests (POCTs) can be performed at or near the point of care.
• They are often performed by clinicians rather than laboratory staff.
• POCTs for infection diagnosis are often based on Immunochromatographic Assays (ICAs).
• POCTs can be efficient and provide rapid results.
• They are often portable and relatively easy to use.
• POCTs can be costly, prone to processing errors and have a higher margin of error than lab methods.

Laboratory Diagnostics: Nucleic Acid Identification Tests

• These tests are highly sensitive and specific.
• They are useful for identifying bacterial, fungal, protozoan, and viral infections.
• They require commercial kits, technical expertise, and expensive equipment.
• Examples of NAATs include PCR and CRISPR.

Nucleic Acid Identification Tests: Polymerase Chain Reaction (PCR)

• PCR is highly sensitive, capable of detecting a single cell or virion.
• It amplifies target sequences, creating billions of copies in a few hours.
• It is highly specific, targeting only DNA that perfectly matches primers.

Nucleic Acid Identification Tests: DNA Microarrays

• DNA microarrays are based on the ability of single-stranded DNA (ssDNA) to bind to its complementary sequence.
• They utilize short ssDNA molecules bound to a glass slide, where complementary DNA molecules in a sample can bind and be visualized.
• They require commercial kits and specialized equipment.
• They are useful for panel tests, such as blood culture panels and respiratory panels.

Common Diagnostic Methods By Microbe Type

• Bacteria:
  • Traditional culture
  • Microscopy
  • Immunological methods
  • Nucleic acid tests, especially for bacteremia, meningitis, and hard-to-culture species
• Viruses:
  • Nucleic acid tests
  • Serology (antibody detection)
• Yeasts/Molds:
  • Traditional culture
  • Microscopy
  • Serology (antibody detection)
• Protozoa:
  • Microscopy
  • Immunological methods are rare (ICA for malaria only)
  • Nucleic acid tests are rare (enteric panel)
• Helminths:
  • Microscopy

Sensitivity vs Specificity

• Both sensitivity and specificity measure a test's ability to correctly classify a person as having or not having a disease.
• Sensitivity refers to a test's ability to correctly identify individuals with the disease (few false negatives).
• Specificity refers to a test's ability to correctly identify individuals without the disease (few false positives).

Laboratory Diagnostics: Overview

• Infectious diseases are diagnosed through methods like culturing pathogens, microscopic identification, detecting antibodies or antigens, or detecting pathogen DNA or RNA.
• Laboratory diagnostic methods include culture methods, microscopy, immunological methods, and molecular methods.
• Each method has a unique set of advantages and disadvantages.
• Different methods are appropriate for different types of infections.

Sensitivity and Specificity

• Sensitivity: A test’s ability to correctly identify individuals with a disease.
• A highly sensitive test has few false negatives, meaning fewer cases of the disease are missed.
• Specificity: A test's ability to correctly identify individuals without a disease.
• A highly specific test has few false positives, meaning there are fewer incorrect diagnoses.

Methods of Identifying Microorganisms

• Morphology: Assessing the size, shape, and arrangement of microorganisms under a microscope.
• Biochemical Tests: Determining the presence of specific enzymes in a microorganism.
• Immunological Tests: Utilizing antibodies to detect specific proteins in patient samples.
• Nucleic Acid Tests: Detecting the presence of microbial DNA or RNA in patient samples.

Immunological Methods

• Common: Agglutination reactions, EIAs/ELISAs, Immunochromatography assays (ICAs)
• Less common: Immunoprecipitations, Neutralization, Interferon gamma release assays (IGRAs), Western blotting, and complement fixation.

Agglutination Reactions

• Occur when antibodies bind antigens, forming clumps.
• Latex agglutination is a common method using synthetic beads coated with antigens.
• Used for blood typing and identifying specific bacteria like Staphylococcus aureus.

Enzyme Immunoassays (EIAs) or Enzyme-linked Immunosorbent Assays (ELISAs)

• Use enzyme-bound antibodies to detect antigens in a sample.
• Typically use a chromogen that changes color as a result of an enzymatic reaction.

Point-of-Care Tests (POCTs)

• Diagnostic tests performed at or near the point of care, often by clinicians.
• Examples: Strep throat tests, Trichomonas vaginalis tests, urine pregnancy tests.
• Pros: Efficiency, speed, portability, ease of use.
• Cons: Cost, potential for processing errors, lower sensitivity and specificity compared to laboratory methods.

Nucleic Acid Identification Tests

• Highly sensitive and specific.
• Common methods: Polymerase chain reaction (PCR) and DNA microarrays.
• Less common methods: CRISPR, Genome mapping.
• Requires specialized equipment and technical expertise.

Polymerase Chain Reaction (PCR)

• Amplifies DNA or RNA sequences in a sample, detecting even single cells or virions.
• Highly specific, targeting only specific DNA sequences.
• Applications include diagnosing infections and facilitating genetic research.

DNA Microarrays

• Based on the ability of single-stranded DNA (ssDNA) to bind to its complementary sequence.
• Detect a variety of microbes by targeting multiple genes/sequences, creating a comprehensive molecular profile.
• Used in blood culture panels and respiratory panels.

Common Diagnostic Methods for Different Microbes

• Bacteria: Traditional culture, microscopy, immunological methods, nucleic acid tests (especially for bacteremia, meningitis, and difficult-to-culture species).
• Viruses: Nucleic acid tests, serology (antibody detection).
• Yeasts/Molds: Traditional culture, microscopy, serology (antibody detection).
• Protozoa: Microscopy, immunological methods (ICA for malaria only), nucleic acid tests (rare, used in enteric panels).
• Helminths: Microscopy.

This quiz explores Point-of-Care Tests (POCTs) and Nucleic Acid Identification Tests (NAATs), including PCR. Gain insights into the advantages and limitations of these diagnostic methods. Test your knowledge on how these innovative techniques aid in the rapid diagnosis of infections.