Molecular Assay (Qualitative and Quantitative) of HIV
المؤلف:
Baijayantimala Mishra
المصدر:
Textbook of Medical Virology
الجزء والصفحة:
2nd Edition , p264-266
2025-12-10
118
The molecular assays used for the diagnosis of HIV can be broadly divided into two types— qualitative assay and quantitative assay.
Qualitative assays: These assays are used either for detection of HIV RNA or for detection of HIV proviral DNA. Reverse transcriptase polymerase chain reaction (RT PCR) is the most commonly used molecular technique for this purpose. However, other methods such as branched DNA (b-DNA), nucleic acid sequence based assay (NASBA) and transcription mediated amplification (TMA) are also used but less commonly than RT-PCR. Roche, Abbott, BioMeriux and Siemens are the leading manufacturer of HIV RT-PCR. All are based on real-time PCR plat form except Siemens’ Versant HIV-1 which is based on signal amplification principle of branched DNA assay. The dynamic range of detection varies from <100 copies/mL to 108 copies/mL.
Indication of Qualitative HIV PCR:
• Screening of blood donors (NAAT: Nucleic acid amplification technique),
• During the window period particularly when p24 antigen is negative,
• Diagnosis in newborns born to HIV positive mothers, in children up to the age of 18 months (due to the possibility of maternal HIV antibody).
• To diagnose suspected acute HIV infection.
• In case of indeterminate serological test
HIV proviral DNA: This is an integrated form of HIV DNA with the host cell chromo some and is a sensitive marker to establish the infection. This cell associated viral DNA is detected from peripheral blood mononuclear cells (PBMC). The detection is helpful in diagnosis in infants as well as after antiretroviral treatment (ART).
Point of care test (POCT) for HIV PCR: WHO in its updated recommendation in 2021 on “HIV prevention, infant diagnosis, antiretroviral initiation and monitoring”, strongly recommends the use of point of care molecular tests (POCT) for diagnosis in infants and children <18 months in order to enable early initiation of ART and to reduce the mortality. Two POCT platforms have so far been approved for HIV diagnosis; Abbott m-PIMATM and Cepheid GeneXPert®.
Causes of false negative HIV PCR: Though PCR is a highly sensitive and specific test, false negative result can occur: (i) In case of HIV-2, if it is not included in the target, (ii) after antiretroviral therapy because of decrease in viral load below the detection limit, (iii) infection with a new variant HIV, or (iv) in “elite controller” in whom the viral replication may remain suppressed for a long period even without ART.
Quantitative assay: Determination of viral load is an essential tool used for predicting the disease course as well as for monitoring the response to antiretroviral therapy.
HIV RNA load: The plasma HIV RNA load is a marker of disease progression. Determination of plasma HIV-1 RNA load is used a guiding criterion for the clinicians to start, and monitor the antiretroviral therapy and also to decide the therapeutic strategy. The methods employed are either target amplification technique such as; real-time PCR (qPCR), nucleic acid sequence-based amplification (NASBA) or signal amplification technique like branched- DNA technique (bDNA). These tests can detect up to 20–40 copies of virus/ mL plasma.
Cell associated HIV DNA: Recently the total cell associated HIV DNA that includes both integrated and non-integrated viral genomes is considered as an important marker to measure the cell associated HIV reservoir. It can be measured in whole blood, cell or tissues. The total HIV DNA load can act as a predictor of disease kinetics, helps in therapeutic monitoring in terms of predicting the treatment outcome and also has been found to be useful in guiding the therapeutic strategy. It also helps to study the HIV infection status in lymph nodes and tissues and thus can help to study the effect of antivirals on those sites.
Viral load monitoring: This is an inherent component of HIV patient management. It is performed to: (i) Detect the treatment failure at the earliest, (ii) helps in assessing the response to treatment in terms of predicting the risk of future development of treatment failure, and (iii) risk of HIV transmission.
Timing of viral load testing:
• The first viral load determination should be done before initiation of ART to find the baseline. The subsequent viral load monitoring is done at various time interval. The viral load testing algorithm as prescribed by WHO is given in Fig. 1.
• In patients with first-line ART, viral load testing needs to be done after 6 months, after 12 months in first year and every year annually.
• In patients with second/third line ART, viral load testing every six months.
Fig1. WHO recommended post-ART viral load testing algorithm (Source: Updated recommendations on HIV prevention, infant diagnosis, antiretroviral initiation and monitoring (who.int) WHO. March 2021)
WHO’s criteria for different point of treatment response:
• Treatment failure threshold: ≥1000 copies/ mL, with >95% of treatment adherence for each of the last 3 months.
• Viral suppression and undetectability: ≤ 50 copies/mL.
• Low level viremia: One or more viral load in one person between 50 and 1000 copies/mL. HIV viral load <1000 copies/mL also has been shown not to be associated with transmission.
The relationship between different ranges of viral load among low level viremia is:
• 50–200 copies/mL shows a trend of predicting towards a future development of virological failure.
• 200–500 copies/mL strongly predicts towards a future development of virological failure.
• 500–1000 copies/mL typically predict a future development of virological failure.
Role of POCT in viral load monitoring: Several POCTs have also been approved by WHO for viral load determination; Cepheid Gene XPert® and Abbott m-PIMATM. The pooled sensitivity and pooled specificity of Cepheid GeneXPert® has been found to be 96.5% and 96.6% respectively (>96% both) for a treatment failure threshold of 1000 copies/mL. The sensitivity of Abbott m-PIMATM in two studies carried out in Kenya and Brazil has been reported to be 95.4% and 97.1% (>95% sensitivity), whereas the specificity in these studies was 96% and 76% for a treatment failure threshold of 1000 copies/mL.
In a program-based scenario, viral load testing by POCT is given priority in following scenario (as per WHO’s 2021 recommendation):
• Pregnant and breastfeeding women
• Infants, children and adolescents • People requiring a repeat viral load after a first elevated viral load
• People for whom treatment failure is suspected
• People presenting sick, living with advanced HIV disease or having a known opportunistic infection (TB, cryptococcal infection, etc.)
• First scheduled viral load test for people reentering care.
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