Nucleic Acid Amplification Tests for Enteric Parasites

NAAT - most promising alternative platform for detection of pathogenic parasites in stool specimens

- is a molecular technique used to detect a particular pathogen in a specimen of blood or other

tissue or body fluid
- detecting and amplifying the RNA or DNA of the pathogen by making extra copies of its nucleic
acid

Types:

1. Polymerase Chain Reaction – use a primer to rapidly make copies of the genetic material.

2. Branched DNA (Quantiplex bDNA) – use a molecule that links to the specific genetic material

3. Ligase Chain Reaction – amplifies the nucleic acid used as the probe. Uses DNA polymerase and
DNA ligase.

4. Transcription Mediated Amplification (TMA) – uses a slightly different molecular method than
PCR but has the same basic principle.

5. Nuclei Acid Sequence – Based Amplification – amplify RNA sequences

Used in: SARS, Human Bocavirus, Trypanosoma brucei

6. Loop Mediated Isothermal Amplification (LAMP) – is a single tube technique for the
amplification of DNA.
Used in: Tuberculosis, Malaria, Sleeping Sicknes

NAAT: POLYMERASE CHAIN REACTION IN PARASITOLOGY

- 2013: first commercial kit FDA approved for diagnostic use

Delay in NAAT applications:

1. DNA extraction in stool is complicated by inhibition and cross contamination problems

2. “Fluid handling” instruments often perform poorly with stool matrices

Advantages:

1. Reduce turnaround time and labor costs

2. Syndrome-based microbiological diagnosis
3. Higher analytic sensitivity compared to Microscopy
4. Can differentiate morphologically identical organisms
5. Capable of creating panels that are patient centered
RESULTS AND ANALYSIS:

1. Qualitative PCR - when PCR is used to detect the presence or absence of a specific DNA product
2. Quantitative real-time or qRT-PCR - indicates how much of a specific DNA or gene is present in
the sample.

Two main methods of visualizing the PCR products:

1. staining of the amplified DNA product with a chemical dye such as ethidium bromide.
2. labeling the PCR primers or nucleotides with fluorescent dyes (fluorophores) prior to PCR
amplification