Additional HIV cards Flashcards
Outline the COBAS6800 Viral load procedure (4 steps)
REAL TIME PCR
- Collection of blood specimen, seperation and storage of plasma sample
- Automated HIV RNA isolation
- Automated reverse transcription of target RNA to generate cDNA and PCR amplification of the targets on cDNA.
- Detetion of cleaved dual labelled oligionucleotide probes to specific targets (Taqman probes)
Why is the WEstern blot good for HIV?
- Presentation of banding reflects the stage of disease
- Remains useful supplemental assay due to HIGH SPECIFICITY
- Independent check for potential vertical transmission of HIV
How does REAL TIME PCR work Specifically the 2 detection methods
The entire viral RNA is present in the sample with the target RNA section within the RNA.
- reverse transcription (RT in PCR master mix) converts the HIV RNA into cDNA
2.PCR occurs (denaturation, annealing of primers and probes/dyes, extension)
SYBR Green
SYBR green does not fluroscence when not bound to dsDNA. When dsDNA is present, the SYBR green will bind (target or not, not specific) and will emit flurosence ONLY when bound. The binding of the dye occurs during the EXTENSION PHASE as this is when the ssDNA is converted to dsDNA. the MORE dsDNA made, the MORE fluroscence.
The primers will bind to the target region of the DNA and cause extension of this segment. After each cycle (denature, annealing, extension), the level of fluroscence increases as the amplification of the target DNA is amplified. The fluroscence is measured after each cycle and is compared to the CT value (cycle number at which threshold fluroscence is acheived)
NO target: Nothing for primers to bind so no amplification of DNA, no fluroscence.
Relationship between cycle and fluroscence (assuming virus RNA is present)
Ct is at a set threshold, the cycle number required in order for fluroscence to be detected. The samples with more viral DNA/RNA will require less cycles for the dye or probes to the target and cause fluroscence. More target = more space for detector to bind = more detector bound = more FLUROSCENCE.
Whereas a sample with less virus genome = more cycles needed for enough detector to bind to create a detectable level of fluroscence.
SO a HIGH CT value = LESS genome in sample
LOW CT = HIGH viral load
Principle of Taqman in REAL TIME PCR
During PCR, the forward and reverse primers hybridize to a specific sequence product. A taqman probe which is present in the same reaciton mixture consists of a 5’ reporter dye and a 3’ quencher dye hybridzes to a target sequence in the PCR product. A Taqpolymerase which has a 5’-3’ exonuclease activiity cleaves the probe. The reporter dye and quncher dye are speratered upon cleavage and resutls in fluroscence of the reporter dye. Thus the increase in fluroscence is directly related to the target amplification in the PCR.
Eclipse phase vs acute phase
Eclipse Phase: The eclipse phase refers to the period immediately following HIV transmission, during which the virus has entered the body but is not yet detectable in standard blood tests. This is because it takes time for the virus to establish itself in the body and for the immune system to develop antibodies against it. The length of the eclipse phase can vary from person to person but typically lasts from a few days to a few weeks.
Acute Phase: The acute phase of HIV infection occurs after the eclipse phase. During this phase, the virus replicates rapidly in the body, and the person may experience flu-like symptoms, such as fever, fatigue, rash, and swollen lymph nodes. This phase typically lasts a few weeks, after which the person may enter a clinical latency stage where the virus is still active but at lower levels, and there may be no symptoms for many years
2 reasons why viral load is important for HIV patients
Treatment Monitoring: Viral load indicates the amount of HIV virus present in a person’s blood. Monitoring viral load is crucial to assess the effectiveness of antiretroviral therapy (ART), which is the primary treatment for HIV. The goal of ART is to suppress the viral load to undetectable levels. When the viral load is undetectable, it means that the level of HIV in the blood is so low that it cannot be detected by standard tests. This indicates that the treatment is working effectively. Maintaining an undetectable viral load is associated with better long-term health outcomes and reduces the risk of transmitting the virus to others. If a person’s viral load is not responding to treatment or if it becomes detectable after being undetectable, it may indicate the need for a change in the treatment regimen. This might involve switching to a different combination of antiretroviral drugs to better control the virus. It might suggest drug resistance.
Transmission Risk: High viral loads are associated with a greater risk of HIV transmission. Individuals with high viral loads are more likely to transmit the virus to their sexual partners or through other means, such as sharing needles. Reducing the viral load through effective treatment not only benefits the individual’s health but also reduces the risk of transmitting the virus to others, contributing to HIV prevention efforts.
p24 level across disease stages and what do high and low levels of p24 mean
Antigen and Diagnostic Marker: p24 is an HIV core protein, specifically a viral antigen. It is an integral part of the HIV virus and is found in the virus’s core structure. p24 can be detected in the blood during the early stages of HIV infection, even before antibodies against the virus are produced. This makes it a valuable diagnostic marker.
A high concentration of p24 antigen typically indicates high viral replication in the body. The p24 antigen is a core protein of the HIV virus, and its presence in the blood is a direct reflection of the active replication of the virus. During the acute phase of HIV infection, when the virus is rapidly multiplying in the body, the concentration of p24 antigen in the bloodstream is usually elevated
During the acute phase, which occurs shortly after HIV transmission, the virus replicates rapidly in the body.
The concentration of p24 antigen in the blood is relatively high in this phase, as it reflects the presence of the virus’s core protein.
p24 antigen tests can be used to detect and diagnose HIV infection during the acute phase, often before HIV antibodies become detectable. This makes them valuable tools for early diagnosis.
As the infection progresses and the individual enters the chronic phase, the concentration of p24 antigen in the blood decreases.
The immune system typically starts producing HIV antibodies, which are more commonly used for HIV diagnosis in later stages.
While p24 antigen can still be detected in the blood during chronic infection, its levels tend to decrease, and it becomes less relevant for diagnostic purposes.
In individuals on antiretroviral therapy (ART), the goal is to reduce the viral load, including the levels of p24 antigen, to undetectable or very low levels.
Monitoring p24 levels, along with viral load measurements and CD4 cell counts, is important for assessing the effectiveness of treatment and managing HIV.
PCR vs NAT
NAT is a highly sensitive test used to directly detect the genetic material (RNA or DNA) of the virus, in this case, HIV.
PCR measures the amount of HIV RNA (in the case of HIV-1) in a person’s blood
comparison of acute vs chronic points acronym
4 PITAS AV
CD4 count
Acute: CD4 cell count is typically normal or slightly decreased during acute infection. May not immediately reflect the damage to the immune system
Chronic:CD4 cell count gradually declines over time, indicating immune system weakening. Regular monitoring is crucial to assess immune health
Progression (Immune response related):
Acute:Rapid viral replication and immune response. - Immune system is actively engaged in fighting the virus, which can contribute to symptoms.
Chronic:Gradual weakening of the immune system over time. - Less active viral replication, but the virus is still present in the body
(Opportunistic) infections
Acute: Rare during this stage, as the immune system is still actively fighting the virus. - Some individuals may experience transient skin rashes or mucosal lesions
Chronic: Opportunistic infections may become more common as immune function weakens over time. - Risk of conditions like thrush or oral candidiasis may increase
Transmission risk
Acute:High risk of transmitting the virus to sexual partners or through needle sharing. - Individuals may not be aware of their infection.
Chronic:Lower risk of transmission compared to acute infection. - Transmission risk is still present if precautions are not taken
ART
Acute:Not always initiated during this stage, as symptoms may not be recognized as HIV-related. - Testing for HIV antibodies is usually the primary diagnostic method
Chronic:Generally recommended to manage the infection, control viral replication, and preserve immune function. - ART initiation is based on CD4 cell counts, viral load, and clinical guidelines
Symptoms
Acute:Often presents with flu-like symptoms, such as fever, fatigue, sore throat, rash, swollen lymph nodes.
Chronic: Symptoms can be severe in some cases and may be mistaken for another illness. Typically asymptomatic or mild symptoms. Many don’t have symptoms and experience no symptoms during this stage
AIDS
Acute:AIDS is not typically diagnosed during this stage. - Individuals with acute infection can progress to AIDS if left untreated.
Chronic: AIDS can develop if left untreated and immune function deteriorates.
Diagnosis is based on CD4 cell count (<200) and the presence of specific opportunistic infections or cancers
Viral load
Acute: High viral load in the blood, as the virus replicates rapidly.
High risk of transmitting the virus to others during this stage
Chronic:Symptoms can be severe in some cases and may be mistaken for another illness. Typically asymptomatic or mild symptoms. Many don’t have symptoms and experience no symptoms during this stage
How early can P24 be detected?
The p24 Ag is detectable as early as 14 days after exposure
Testing for HIV at different stages AND timeframe for each stage of HIV
Eclipse: 0-10 days
Acute: 10-30 days (2-4 weeks) (seroconversion around 4-6 weeks)
Chronic: years
Eclipse: No tests
Acute: NAT (HIV RNA) (10- 33days), p24 (15+ days), WB (25+ days), Ag/Ab testing (18-45 days)
Chronic: Ab testing (23-90 days)
when does seroconversion occur
4-6 weeks (late acute)
