Viral load, also known as viral burden, is a numerical expression of the quantity of virus in a given volume of fluid, including biological and environmental specimens. It is not to be confused with viral titre or viral titer, which depends on the assay. When an assay for measuring the infective virus particle is done (Plaque assay, Focus assay), viral titre often refers to the ''concentration'' of infectious viral particles, which is different from the ''total'' viral particles. Viral load is measured using body fluids
Sputum
Sputum is mucus that is coughed up from the lower airways (the trachea and bronchi). In medicine, sputum samples are usually used for a naked eye examination, microbiological investigation of respiratory infections and cytological investigations ...
and
blood plasma
Blood plasma is a light amber-colored liquid component of blood in which blood cells are absent, but contains proteins and other constituents of whole blood in suspension. It makes up about 55% of the body's total blood volume. It is the intra ...
.
[ As an example of environmental specimens, the viral load of ]norovirus
Norovirus, sometimes referred to as the winter vomiting disease, is the most common cause of gastroenteritis. Infection is characterized by non-bloody diarrhea, vomiting, and stomach pain. Fever or headaches may also occur. Symptoms usually deve ...
can be determined from run-off water on garden produce. Norovirus
Norovirus, sometimes referred to as the winter vomiting disease, is the most common cause of gastroenteritis. Infection is characterized by non-bloody diarrhea, vomiting, and stomach pain. Fever or headaches may also occur. Symptoms usually deve ...
has not only prolonged viral shedding
Viral shedding is the expulsion and release of virus progeny following successful reproduction during a host cell infection. Once replication has been completed and the host cell is exhausted of all resources in making viral progeny, the viruse ...
and has the ability to survive in the environment but a minuscule infectious dose is required to produce infection in humans: less than 100 viral particles.
Viral load is often expressed as viral particles, (virions) or infectious particles per mL depending on the type of assay. A higher viral burden, titre, or viral load often correlates with the severity of an active viral infection. The quantity of virus per mL can be calculated by estimating the live amount of virus in an involved fluid. For example, it can be given in RNA
Ribonucleic acid (RNA) is a polymeric molecule essential in various biological roles in coding, decoding, regulation and expression of genes. RNA and deoxyribonucleic acid ( DNA) are nucleic acids. Along with lipids, proteins, and carbohydra ...
copies per millilitre of blood plasma.
Tracking viral load is used to monitor therapy during chronic viral infections, and in immunocompromised patients such as those recovering from bone marrow
Bone marrow is a semi-solid tissue found within the spongy (also known as cancellous) portions of bones. In birds and mammals, bone marrow is the primary site of new blood cell production (or haematopoiesis). It is composed of hematopoietic ce ...
or solid organ transplantation
Organ transplantation is a medical procedure in which an organ (anatomy), organ is removed from one body and placed in the body of a recipient, to replace a damaged or missing organ. The donor and recipient may be at the same location, or organ ...
. Currently, routine testing is available for HIV
The human immunodeficiency viruses (HIV) are two species of ''Lentivirus'' (a subgroup of retrovirus) that infect humans. Over time, they cause acquired immunodeficiency syndrome (AIDS), a condition in which progressive failure of the immune ...
-1, cytomegalovirus
''Cytomegalovirus'' (''CMV'') (from ''cyto-'' 'cell' via Greek - 'container' + 'big, megalo-' + -''virus'' via Latin 'poison') is a genus of viruses in the order ''Herpesvirales'', in the family ''Herpesviridae'', in the subfamily ''Betaherpe ...
, hepatitis B
Hepatitis B is an infectious disease caused by the ''Hepatitis B virus'' (HBV) that affects the liver; it is a type of viral hepatitis. It can cause both acute and chronic infection.
Many people have no symptoms during an initial infection. Fo ...
virus, and hepatitis C
Hepatitis C is an infectious disease caused by the hepatitis C virus (HCV) that primarily affects the liver; it is a type of viral hepatitis. During the initial infection people often have mild or no symptoms. Occasionally a fever, dark urine, a ...
virus. Viral load monitoring for HIV Viral load monitoring for HIV is the regular measurement of the viral load of individual HIV-positive people as part of their personal plan for treatment of HIV/AIDS.
A count of the viral load is routine before the start of HIV treatment.
Types
Va ...
is of particular interest in the treatment of people with HIV
HIV-positive people, seropositive people or people who live with HIV are people who have the human immunodeficiency virus HIV, the agent of the currently incurable disease AIDS.
According to estimates by WHO and UNAIDS, 34.2 million people were ...
, as this is continually discussed in the context of management of HIV/AIDS
The management of HIV/AIDS normally includes the use of multiple antiretroviral drugs as a strategy to control HIV infection. There are several classes of antiretroviral agents that act on different stages of the HIV life-cycle. The use of multiple ...
. An undetectable viral load does not implicate a lack of infection. HIV positive patients on long-term combination antiretroviral therapy may present with an undetectable viral load on most clinical assays since the concentration of virus particles is below the limit of detection
The limit of detection (LOD or LoD) is the lowest signal, or the lowest corresponding quantity to be determined (or extracted) from the signal, that can be observed with a sufficient degree of confidence or statistical significance. However, the ...
(LOD).
Technologies for viral load testing
A 2010 review study by Puren ''et al.'' categorizes viral load testing into three types: (1) nucleic acid
Nucleic acids are biopolymers, macromolecules, essential to all known forms of life. They are composed of nucleotides, which are the monomers made of three components: a 5-carbon sugar, a phosphate group and a nitrogenous base. The two main cl ...
amplification based tests (NAT
Nat or NAT may refer to:
Computing
* Network address translation (NAT), in computer networking
Organizations
* National Actors Theatre, New York City, U.S.
* National AIDS trust, a British charity
* National Archives of Thailand
* National As ...
s or NAATs) commercially available in the United States with Food and Drug Administration
The United States Food and Drug Administration (FDA or US FDA) is a List of United States federal agencies, federal agency of the United States Department of Health and Human Services, Department of Health and Human Services. The FDA is respon ...
(FDA) approval, or on the market in the European Economic Area
The European Economic Area (EEA) was established via the ''Agreement on the European Economic Area'', an international agreement which enables the extension of the European Union's single market to member states of the European Free Trade Ass ...
(EEA) with the CE marking
On commercial products, the letters CE (as the logo ) mean that the manufacturer or importer affirms the good's conformity with European health, safety, and environmental protection standards. It is not a quality indicator or a certificatio ...
; (2) "Home–brew" or in-house NATs; (3) non-nucleic acid-based test.
Nucleic acid-based tests (NATs)
There are many different molecular based test methods for quantifying the viral load using NATs. The starting material for amplification can be used to divide these molecular methods into three groups:
# Target amplification which uses the nucleic acid itself. Just a few of the more common methods
#* The polymerase chain reaction
The polymerase chain reaction (PCR) is a method widely used to rapidly make millions to billions of copies (complete or partial) of a specific DNA sample, allowing scientists to take a very small sample of DNA and amplify it (or a part of it) t ...
(PCR) method of in vitro
''In vitro'' (meaning in glass, or ''in the glass'') studies are performed with microorganisms, cells, or biological molecules outside their normal biological context. Colloquially called "test-tube experiments", these studies in biology an ...
DNA synthesis uses a DNA template, polymerase
A polymerase is an enzyme ( EC 2.7.7.6/7/19/48/49) that synthesizes long chains of polymers or nucleic acids. DNA polymerase and RNA polymerase are used to assemble DNA and RNA molecules, respectively, by copying a DNA template strand using base- ...
, buffers, primers, and nucleotides
Nucleotides are organic molecules consisting of a nucleoside and a phosphate. They serve as monomeric units of the nucleic acid polymers – deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), both of which are essential biomolecules w ...
to multiply the HIV in the blood sample. Then a chemical reaction marks the virus. The markers are measured and used to calculate the amount of virus. PCR is used to quantify integrated DNA.
#* Reverse transcription polymerase chain reaction
Reverse transcription polymerase chain reaction (RT-PCR) is a laboratory technique combining reverse transcription of RNA into DNA (in this context called complementary DNA or cDNA) and amplification of specific DNA targets using polymerase cha ...
(RT-PCR) is a variation of PCR that can be used to quantify viral RNA. RNA is used as the starting material for this method and converted to double-stranded DNA, using the enzyme reverse transcriptase
A reverse transcriptase (RT) is an enzyme used to generate complementary DNA (cDNA) from an RNA template, a process termed reverse transcription. Reverse transcriptases are used by viruses such as HIV and hepatitis B to replicate their genomes, ...
(RT) for PCR.
#* The nucleic acid sequence-based amplification (NASBA) method is a transcription-based amplification system (TAS) variation of PCR. RNA is used as the target and a DNA copy is made. The DNA copy is then transcribed into RNA and amplified. Several TAS commercial variations are available including; transcription-mediated amplification (TMA), and self-sustaining sequence replication (3SR).
# Probe specific amplification uses synthetic probes that preferentially bind to a target sequence. The probes are then amplified
# Signal amplification uses large amounts of signal bound to an unamplified target originally present in the sample. One commonly used method:
#* The branched DNA
In biology, a branched DNA assay is a signal amplification assay (as opposed to a target amplification assay) that is used to detect nucleic acid molecules.
Method
A branched DNA assay begins with a dish or some other solid support (e.g., a plast ...
(bDNA) method can use either DNA or RNA as the target nucleic acid. Short probes attached to a solid support and capture the target nucleic acid. Additional extender probes also bind to the target nucleic acid and to numerous reporter molecules which are used to increase the signal intensity, which is converted to a viral count.
Plasma specimens
EDTA
Ethylenediaminetetraacetic acid (EDTA) is an aminopolycarboxylic acid with the formula H2N(CH2CO2H)2sub>2. This white, water-soluble solid is widely used to bind to iron (Fe2+/Fe3+) and calcium ions (Ca2+), forming water-soluble complexes eve ...
plasma is a good source of cell-free viral RNA for RNA-based viral load testing. Consideration of specimen
Specimen may refer to:
Science and technology
* Sample (material), a limited quantity of something which is intended to be similar to and represent a larger amount
* Biological specimen or biospecimen, an organic specimen held by a biorepository ...
collection, storage and biosafety
Biosafety is the prevention of large-scale loss of biological integrity, focusing both on ecology and human health.
These prevention mechanisms include conduction of regular reviews of the biosafety in laboratory settings, as well as strict guidel ...
measures is essential. Extraction of RNA from plasma requires specialized equipment, reagents
In chemistry, a reagent ( ) or analytical reagent is a substance or compound added to a system to cause a chemical reaction, or test if one occurs. The terms ''reactant'' and ''reagent'' are often used interchangeably, but reactant specifies a ...
and training, placing it out of reach for medium to small labs with limited resources. A large sample (> 1 mL of plasma) is needed for a linear range bottoming out at 50 copies/mL, requiring venipuncture
In medicine, venipuncture or venepuncture is the process of obtaining intravenous access for the purpose of venous blood sampling (also called ''phlebotomy'') or intravenous therapy. In healthcare, this procedure is performed by medical labo ...
. This linear range is best for treatment monitoring. If a higher linear range of more than 1000 copies/mL is acceptable, a finger stick
In medicine, some blood tests are conducted on capillary blood obtained by fingerstick (or fingerprick) (or, for neonates, by an analogous heelprick). The site, free of surface arterial flow, where the blood is to be collected is sterilized with a ...
would supply a sufficient specimen for diagnosis of HIV infection during infancy.
Storage
EDTA plasma can be stored at room temperature for 30 hours, 14 days at 4 °C and extended periods of time at -70 °C without significant decreases in viral load signal. The RNA in smaller blood specimens, such as dried plasma spots (DPS) or dried blood spots (DBS) from finger sticks, is reportedly stable at room temperature for periods ranging from 4 weeks to 1 year. The virus is inactivated in dried samples, reducing the danger from specimen handling. DBS and DPS were successfully evaluated for viral load testing, but their linear range is 3 log10 or 4 log10 copies/mL. Because of this lack of sensitivity, dried specimens are useful for HIV screening but not for viral load determination.
Measuring
Viral load is typically reported as copies of HIV in a milliliter (mL) of blood. Changes in viral load are usually reported as a log change (in powers of 10). For example, a three log increase in viral load (3 log10) is an increase of 103 or 1,000 times the previously reported level, while a drop from 500,000 to 500 copies would be a three-log-drop (also 3 log10).
Other factors that affect viral load
Different test methods often give different results for the same patient sample. To be comparable the same test method (target amplification, probe specific amplification, or signal amplification) should be used each time a patient specimen is run. Ideally patient testing should be conducted at the same medical laboratory, using the same viral load test and analyzer.
Time of day, fatigue, and stress can also affect viral load values. Recent immunizations or infections can affect the viral load test. Testing should be postponed for at least four weeks after an immunization or infection.
References
{{DEFAULTSORT:Viral Load
Virology
HIV/AIDS
Medical tests
Epidemiology
Global health
Infectious diseases
Pandemics
Zoonoses
it:Carica virale