From the Spring 2021 issue of "Microcosm."

The diagnostic modalities depicted here represent simplified versions of key laboratory and point-of-care techniques used to identify and characterize microorganisms from patient samples and/or culture isolates. In general, these tests rely on the detection of specific nucleic acid sequences or proteins for diagnosis. Platform variations and examples have been included.

Immunofluorescence Assays (IFA)

Input: Patient Sample

How it works: Sample is mixed with fluorescently labeled primary antibody that binds a particular antigen (direct) OR with unlabeled antigen and then fluorescently labeled secondary antibody that binds the tail (Fc) region of patient antibodies (indirect). Fluorescence is then detected by microscopy.

Examples: Rabies virus   |   Rickettsia rickettsii

Enzyme Immunoassays (EIA)

Input: Patient Sample

How it works: Direct and indirect EIAs are analogous to direct and indirect IFAs: The primary or secondary antibody is enzyme-linked instead of fluorescently labeled. Substrate for the enzyme is added, and the amount of chromogenic or fluorescent product is measured by spectrophotometer.

Sandwich EIAs use immobilized, unlabeled antibody to bind antigen from a patient sample, then an enzyme-linked form of the same antibody for detection. In competitive EIAs, a patient sample is mixed with enzyme-linked antibody, then added to immobilized antigen. Conversion of the enzyme’s substrate detects the amount of primary antibody captured by the immobilized antigen, which inversely correlates with the amount of antigen in the patient sample.

Examples: Hepatitis C virus  |  Legionella pneumophila serogroup 1

Lateral Flow Immunoassays

Input: Patient Sample

How it works: Sample is drawn by capillary action through a series of overlapping components, including conjugate pad, test and control lines. As it flows laterally through the assay, the analyte mixes with freeze-dried bioactive particles, called conjugates, that have immobilized antibodies (or antigens) displayed on their surfaces.

If the target molecule is present in the sample, it will bind to its chemical partner in the conjugate pad.

In sandwich lateral-flow assays, the test line also contains antibodies that are specific to the target analyte. If present, that analyte will also become associated with or bind to the molecules in the test line and display a visible signal (often indicated by color change or fluorescence).

Competitive lateral-flow assays are generally used to detect smaller analytes that have fewer binding sites. Copies of the target analyte are immobilized in the test line. Only when the analyte is absent from the sample will the conjugate antibodies bind and display a visual signal in the test line.

In both types of assays, the control line shows whether the sample has flowed through the assay and the biomolecules are active.

Examples: Rhinovirus, PIV and Influenza virus rapid antigen detection | Epstein-Barr serology

Nucleic Acid Amplification Tests (NAAT)

Input: Nucleic Acid

How it works: Primers for one or more genomic sequences are used to amplify nucleic acids from a pathogen or group of pathogens. Amplification products are then detected using fluorescent dye, sequence-specific labeled probes or electrophoresis.

Examples: Respiratory virus panels | HIV, HSV, Chlamydia trachomatis and Neisseria gonorrhoeae

Next Generation Sequencing (NGS)

Input: Nucleic Acid

How it works: Various technologies capture all nucleic acid sequences of organisms present in a sample. Data can be analyzed for full genomes of a predominant pathogen (i.e., typing or outbreak-tracing) or a mix of pathogens.

Examples: Genomic surveillance and outbreak identification | Detection of foodborne pathogens

Spectroscopy/Spectrometry

Input: Culture Isolates

How it works: An isolate is either bombarded with infrared light (FTIR) or ionized and run through a detector (MALDI-ToF) to capture a fingerprint-like spectrum. Comparison to a database of known spectra enables organism or strain identification.

Examples: Candida auris | Enterobacter cloacae complex typing