Assays Methods for detecting viral antigens and antibodies in the body fluids of HIV-infected patients are easy to perform and easy to apply universally, with antibody testing being particularly common. However, the status and importance of HIV P24 antigen and viral gene assays in the detection of HIV infection is also receiving increasing attention.
Antibody testing HIV antibody in serum is an indirect indicator of HIV infection. According to its main scope of application, the existing HIV antibody testing methods can be divided into screening tests and confirmatory tests.
Confirmatory reagents The most commonly used confirmatory test for positive sera from screening tests is Western blot (WB), which is only suitable as a confirmatory test due to its relatively long window period, slightly poor sensitivity, and high cost. The other FDA-approved screening test is the immunofluorescence assay (IFA), which is less expensive than the WB and relatively simple to perform, with the entire process completed in 1-1.5 hours. The main disadvantage of this method is that it requires an expensive fluorescence detector and experienced professionals to observe and judge the results, and the results cannot be stored for a long time. The FDA now recommends a negative or positive IFA when releasing final results to blood donors whose WB cannot be determined, but not as a criterion for blood eligibility.
Screening test Screening test is mainly used to screen blood donors, so it requires easy operation, low cost, and sensitive and specific. 2012, the world’s main screening method is still ELISA, there are a few particle agglutination reagents and rapid ELISA reagents. ELISA has high sensitivity and specificity, simple operation, only need laboratory equipped with enzyme marker and plate washer can be It is particularly suitable for use in large scale screening in laboratories.
Particle agglutination test is another simple and convenient operation, low-cost detection method, the results of the method can be determined by the naked eye, high sensitivity, especially suitable for developing countries or a large number of screening blood donors, the disadvantage is that fresh samples must be used, the specificity is poor.
Spot-blot assay (Dot-blot assay) developed in the late 80’s is a rapid ELISA (Rapid ELISA) method, which is extremely easy to operate, the process is short, most of the process can be completed within 5-10 minutes or even 3 minutes, but the method is much more expensive than ELISA and particle agglutination reagents.
Human immunodeficiency virus antibody oral mucosal exudate test kit (colloidal gold method) belongs to the category of lateral immunochromatography (gold immunoassay), which is a rapid diagnostic reagent based on immunochromatographic technology that can be used to detect HIV-1 and HIV-2 antibodies in oral mucosal exudate samples by manual operation, reading the results with the naked eye, and qualitative results in 20 minutes. It can be used for voluntary counseling tests, reluctant blood collection, and primary screening of patients with needle dizziness. The method is suitable for primary screening tests, and anyone who is determined positive by this reagent needs to undergo further screening for confirmation.
HIV negative] means that HIV antibodies are not detected from the human body, and the negative symbol is indicated by (-). It depends on when the test was done. During the window period, the infected person’s body has not yet produced HIV antibodies, or has not yet produced a sufficient amount of HIV antibodies, when the HIV test is negative, if the test is done after the window period, the possibility of HIV infection can be ruled out.
[HIV positive] indicates that HIV antibodies are detected from the human body, and the positive symbol is indicated by (+).
Factors of indeterminate test results Infection is still in the window period: the period from HIV entering the body to the test is not long enough, so the serum has not yet formed a typical antibody response AIDS progresses to the end stage, the antibody level decreases Cross-reaction of other non-viral protein antibodies: autoimmune diseases, certain malignant diseases, pregnancy, blood transfusion or organ transplantation, etc., the body can produce some antibodies whose reaction is similar to The reaction caused by HIV P24 core protein antibodies is very similar.
Antigen detection Pathogen detection mainly refers to the direct detection of virus or virus genes from host specimens by viral isolation and culture, electron microscopic morphological observation, viral antigen detection and genetic assay. Because the first two methods are difficult and require special equipment and specialized technicians. Therefore, only antigen detection and RT-PCR (reverse transcription-PCR) can be used for clinical diagnosis. HIV-1P24 antigen detection can be used as an aid in the diagnosis of HIV-1 antibody uncertainty or window period; an aid in the early differential diagnosis of infants born to HIV-1 antibody-positive mothers; a positive HIV-1 antigen/antibody ELISA reagent test of the fourth generation, but The P24 antigen test is generally performed with ELISA double antibody sandwich reagents, which must be approved and registered by the SDA, and the positive result must be confirmed by neutralization test according to the reagent instructions. HIV-1 P24 antigen test negative only means no reaction in this test, and cannot exclude HIV infection, so it is generally not used as a routine diagnostic item in clinical practice.
HIV nucleic acid testing can be used to assist in the diagnosis of HIV infection, monitor the course of the disease, guide the treatment plan and determine the efficacy, and predict disease progression. Commonly used HIV viral load testing methods include reverse transcription PCR assays (RT-PCR), nucleic acid sequence amplification assays (NASBA), branching DNA hybridization assays (bDNA), and real-time fluorescent quantitative PCR techniques. It is worth noting that each HIV RNA quantification system has its minimum detection limit, i.e., the lowest copy number or international unit that can be measured. A positive HIV nucleic acid test can be used as an auxiliary indicator for the diagnosis of HIV infection and cannot be used for the diagnosis of HIV infection alone. When reporting quantitative HIV nucleic acid test results, the results should be reported according to the instrument readings, indicating the experimental method used, sample type and sample volume, and when the measurement result is less than the minimum detection limit, the minimum detection limit level should be indicated.
Qualitative HIV nucleic acid testing can also be used as an aid in the diagnosis of HIV infection and in basic research such as the analysis of HIV genetic subtypes and variants. PCR or RT-PCR techniques are usually used, using amplification reagents common to molecular biology laboratories. Primers may be derived from the literature or designed by the patient, and should try to cover all or common strains, or a combination of primers may be used. The reaction conditions and the primer sequences used should be indicated when reporting the results of the qualitative assay. In addition, taking advantage of the high sensitivity of nucleic acid detection methods, the use of pooled nucleic acid amplification assays and methods for pooled nucleic acid testing of samples from populations with high suspicion of infection and negative antibodies allows timely detection of window-phase infections. This method is more cost effective than single sample nucleic acid testing.
Culture methods The common method is co-culture, in which single nucleated cells are isolated from normal human peripheral blood, stimulated and cultured with PHA, and then added to the patient’s single nucleated cells for diagnosis and AIDS studies.
After the patient’s own peripheral or bone marrow lymphocytes are stimulated with PHA for 48-72 hours and cultured in vitro (IL2 is added to the culture medium) for 1 to 2 weeks, the virus proliferation can be released into the extracellular space and the cells fuse into multinucleated giant cells, which eventually break down and die. The cells can also be isolated and passaged using passaged lymphocyte lines such as HT-H9 and Molt-4 cells.
The range of HIV infection in animals is narrow, only chimpanzees and gibbons, and generally chimpanzees are used for experiments. Infection of chimpanzees with HIV cells or cell-free HIV filtrate, or transfusion of HIV-infected chimpanzee blood to normal chimpanzees were successful, and HIV was consistently isolated in blood and lymphatic fluid for 8 months, and HIV-specific antibodies were detected after 3-5 weeks and continued to be maintained at a certain level. However, no disease occurred in either chimpanzees or gibbons after infection.