Nucleic acid testing or nucleic acid amplification testing, often abbreviated as NAT or NAAT,

is a technique that involves amplification and detection of genetic material—the nucleic

acids, DNA or RNA—for diagnosis or to provide guidance on therapy. Though the genetic
material of every living being is composed of DNA or RNA, variations exist in genome
sequences. This genetic variation makes NAT an ideal technique for identification of
infectious diseases, cancer, genetic disorders, and mitochondrial disorders and as an aid to
personalized and precision medicine based on the knowledge of pharmacogenomics.

Donors of blood and plasma are tested for antibodies to HCV and antibodies to HIV.
However, there is still a “window period” during which a donor can be infected but have
negative results on these screening tests. With the use of NAT for HCV, the window
Hepatitis B Quantitative Nucleic Acid Amplified Test (NAAT)
Mnemonic HEPBNAATQT
CPT 87517
Test Code 7000010
Synonyms Hep B Quantitative Viral Load, HBV Quantitative Viral Load
Test Includes
Specimen Serum or Plasma
Volume 2.5 mL
Container 3 DEDICATED Lavender Top (EDTA), SST, or Red Top Tubes (These
tubes cannot be used for other testing)
Special Instructions If other tests are requested, additional tubes must be drawn.
Specimen Prep Serum or Plasma must be removed from cells within 24 hours. Centrifuge*
within 24 hours and immediately transfer plasma or serum from all three
Lavender-top, SST, or Red-top tubes to one Purple Screw Cap Tube. BE
VERY CAREFUL NOT TO TRANSFER ANY BLOOD CELLS TO THIS
TUBE. Deliver this tube to the laboratory at room temperature within 24
hours or refrigerated within 3 days.
Storage Refrigerate
Transport Temp Room Temperature
Stability Rm Temp 3 days
Stability Refrig 3 days
Stability Frozen For longer storage, freeze at -20° or below. Once frozen, do not allow to
thaw.
Methodology Polymerase Chain Reaction (PCR)
Rejection Criteria Specimen not processed within 24 hours from time of collection. Hemolyzed
specimen. Specimen collected in Green-top Heparin tube. Delay in transport.
Reference Value Target not detected, <116 copies/mL, <2.1 Log10 copies/mL, <20
International Units (IU)/mL, and <1.3 Log10 IU/mL.
Performed One time per week
Comments The dynamic range of this assay is 20 IU/mL – 170,000,000 IU/mL (116 -
989,400,000 copies/mL).; A result of "Target not detected" means that HBV
RNA was not detected. This result does not rule out HBV infection, as virus
may be present, but below the detectable limit of quantitation.; A result of
<20 IU/mL means that the HBV RNA was detected, but under 20 IU/mL.;
Reported in copies/mL, in Log10 copies/mL, in International Units (IU)/mL,
and in Log10 IU/mL.; Assay results expressed in IU/mL are directly
comparable with results from other assay methods.

period is reduced approximately 50 days (from an average of 57 days to 7 days). For

HIV-1, the average window period with antibody testing is 22 days. This window period
is reduced approximately 12 days with NAT (from an average of 22 days to 10 days).

NAT can be performed on various types of clinical specimens depending on the disease
being diagnosed. Blood, plasma, serum, cerebrospinal fluid, sterile body fluids, sputum,
urine, stool, and tissues are some specimens commonly used for NAT. The nucleic acids are
first extracted from the cell and other cellular components either by manual or automated
methods. The extracted nucleic acids can then be amplified and detected or sequences read
using different methods—polymerase chain reaction (PCR), real-time PCR, microarrays,
sequencing (Sanger and next-generation), etc. With the advancement in the technology for
amplification and detection or sequencing, there has been a great transformation in the
applications of NAT. The major applications are discussed below.

NAT has been associated with blood screening for some time. It was first introduced by the
German Red Cross in 1997 for blood screening to reduce the risk of transfusion-transmitted
viral infections due to the failure of serologic screening tests to detect recently infected
donors in the pre-seroconversion “window” phase of infection. With serology tests, it takes
1

about two months after infection for hepatitis C virus (HCV) antibodies to be detected, while
NAT can detect HCV RNA about five days after infection. To reduce the window period for
detection, European Union regulators began to require in 1999 that all plasma be tested by
NAT techniques for HCV if derivatives made from such plasma were to be sold in Europe.

This announcement was a major impetus for the development and implementation of NAT
of blood and plasma from donors in the United States and other developed countries. In the
U.S., NAT was initially used to screen HCV and human immunodeficiency viruses types 1 and
2 (HIV 1, 2) in blood and blood products, and subsequently extended to hepatitis B virus
(HBV) and West Nile virus (WNV). Screening for human T-lymph tropic virus types I and II
(HTLV-I, II) and Treponema pallidum (causing syphilis) are also routinely performed using
the serological method only. NAT has been adopted in several other countries around the
world, including Canada, France, Australia, New Zealand, South Africa, and other countries in
Europe and Asia. In addition to screening for the organisms mentioned above, blood
2,3

screening for other organisms, including Plasmodium vivax and Plasmodium falciparum
(causing malaria), and Trypanosoma cruzi (causing Chagas disease) are also recommended,
depending on the local epidemiological evidence of the disease.
NAT in detecting infectious diseases
NAT is extensively used to detect and identify organisms for proper diagnosis, prognosis,
and treatment of diseases. Diagnosing infectious diseases by detecting the nucleic acid of
the causative agent in clinical specimens has been found to be more rapid, sensitive, and
specific compared to the traditional method of diagnosis using culture or immunological
methods. Table 1 provides some examples of organisms that have been detected by NAT in
clinical laboratories. 5

Table 1.
Examples of organisms detected by NAT in clinical laboratories.
In virology, in addition to detecting the viruses, NAT further aids in identifying genotypes
and subtypes, determining resistance to particular antibiotics, and measuring viral load.
These applications help to provide guidance on treatment. In bacteriology, NAT has also
been applied to resistance testing, the detection of infection due to fastidious bacteria, and
the detection of bacterial infection after antibiotics have been administered.

NAT also helps in epidemiological studies and infection control. Table 2 provides some
examples of applications of NAT other than detection. 5

Table 2.
Examples of NAT applications other than detection of organisms.

NAT in predicting cancer and guiding cancer treatment

Cancer is a disease caused by an uncontrolled division of abnormal cells in a part of the
body. With improved sequencing capability and knowledge of the human genome (DNA),
traditional descriptions of cancer—for example, by the organ of primary occurrence—are
being superseded by a new classification framework that focuses on the genetic
abnormalities and molecular derangements of malignant tumors. The genetic-level changes
may consist of single or multiple sequence changes in cell DNA (mutations), addition or
deletion of DNA, or changes in the number of copies of particular DNA sequences in cancer
cells. Nucleic acid testing helps to identify genetic variations and predicts predisposition to
cancer, alters diagnostic categories, enhances treatment strategies, enables early detection
and prevention, and improves outcomes for cancer patients. Nucleic acid testing has led to
the emergence of precision and personalized medicine—that is, the tailoring of treatment
based on the individual’s genetic make-up.
Table 3 provides a partial list of predisposition to certain type of cancer based on gene
mutations. NAT helps in detection of the mutations in the genes. 6

Table 3.
Predisposition to type of cancer based on gene mutation.
Table 4 provides a partial list of cancer drugs that are indicated for cancer patients with
particular genetic variants. NAT helps in detection of the patient’s genetic mutations. 7

Table 4.
Cancer drugs indicated for patients with particular mutations.

NAT for personalized and precision medicine

With today’s increased knowledge of pharmacogenomics, nucleic acid testing is being
increasingly used to provide personalized and precision medicine and avoid adverse
reactions from drugs in individuals.

To define the terms: pharmacogenetics studies the effect of a single gene on drug response,
while pharmacogenomics deals with the effects of multiple genes on drug response. The
term pharmacogenetics was coined by Vogel in 1959, but most of the progress in
8

pharmacogenetics has been made in recent years. Researchers and clinicians have come to
understand that every individual metabolizes drugs according to his or her genetic variants
and therefore responds to drugs variably. This has enabled advancements in personalized
and precision medicine.

Over the last decade, the U.S. Food and Drug Administration (FDA) has been aggressive in
providing genetic labeling on new drugs, and also updating product labels for a number of
existing therapies, such as warfarin and 6-mercaptopurine. At present, some 120 drugs have9

pharmacogenetics information in their FDA product label. A current listing of all drugs with
such information in the product label can be found in U.S. FDA Table of Pharmacogenomics
Biomarkers in Drug Labels. Pharmacogenetics information in product labels ranges from
10

boxed warnings, the highest level of warning in the product label, to information in the
clinical pharmacology section.

The Clinical Pharmacogenetics Implementation Consortium (CPIC), formed in 2009, sets

guidelines on drug therapy based on pharmacogenetics information. By early 2013, eight
11

CPIC guidelines had been published, which include TPMT and thiopurines, CYP2C19 and
12

clopidogrel, VKORC1/CYP2C9 and warfarin, CYP2D6 and codeine, HLA-B and abacavir,
13 14 15 16

SLCO1B1 and simvastatin, HLA-B and allopurinol, and CYP2D6/CYP2C19 and tricyclic
17 18

antidepressants. These guidelines are summarized in Table 5 . Production of a number of

19

other guidelines is ongoing and a listing of in-progress guidelines can be found at The
Pharmacogenomics Knowledgebase. 20
 Table 5. Summary of
Clinical Pharmacogenetics Implementation Consortium guidelines.

NAT in screening for genetic disorders

NAT is being extensively used for prenatal screening and carrier screening for genetic
disorders. Congenital anomalies account for 276,000 perinatal deaths by pregnancy Week 4
annually on a global basis. The aim of prenatal screening is to detect birth defects, such as
21

neural tube defects; chromosome abnormalities (e.g., Down syndrome, fragile X syndrome);
and genetic disorders and other conditions (e.g., spina bifida, cleft palate, Tay Sachs disease,
sickle cell anemia, thalassemia, cystic fibrosis, and muscular dystrophy). 21

Carrier screening, the testing of parents in preparation for pregnancy, is used to identify
genetic mutations that could cause serious inherited disorders. Some of the more common
disorders for which such screening is done are cystic fibrosis, sickle cell disease, thalassemia,
and Tay-Sachs disease. 21

In the Ashkenazi (Eastern European descent) Jewish population, a number of genetic

disorders occur more frequently than in the general population. It has been estimated that
one in four individuals is a carrier of one of several genetic conditions. These diseases
include Tay-Sachs disease, Canavan, Niemann-Pick, Gaucher, familial dysautonomia, Bloom
syndrome, Fanconi anemia, cystic fibrosis, and mucolipidosis IV. Table 6 provides a list of
common genetic disorders.
 Table 6. List of common genetic
disorders .
NAT in diagnosis of mitochondrial diseases
Mitochondrial diseases are a group of disorders caused by dysfunctional mitochondria—the
cellular organelle in which respiration and energy formation occur. Mitochondrial diseases
can be caused by genetic mutation in the mitochondrial DNA (mtDNA) or in the nuclear
DNA. Mitochondrial diseases may affect a single organ—for example, the eye in Leber
hereditary optic neuropathy (LHON)—or may involve many organs, and they often result in
major neurologic and myopathic symptoms. Table 7 lists a few disorders caused due to
mutations in mitochondrial DNA. 22