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.