Part 4 Laboratory analysis of HIV infection

After infecting human body, HIV begins to replicate in great number, and can be separated from peripheral blood cells, cerebrospinal fluid and bone marrow fluid. The organism can have different cell immune and body fluid reaction to HIV at different stage of HIV infection, and to confirm the HIV infection and clinical stage, it should be based on laboratory analysis.
To examine HIV infection, any serologic test should have a high sensibility and specificity; however, no reagent can reach 100% accuracy of analysis. The patients with HIV-positive, therefore, should be analyzed once again with a series of experiments available for HIV antigen and antibody test.
1.How to conduct HIV antibody test? Antibody test is a kind of indirect test to confirm the existence of infection through the discovery of antibodies, but not able to finally confirm the existence of viruses. It needs about 3 months from HIV infection to the formation of antibodies, and therefore, this type of test can not identify the infected patient before the formation of antibodies, namely in a “window period”. Both false positive rate and false negative rate of antibody test are very low, with accuracy over 99%. In addition, the test cost is very low, and therefore, it can be applied in the supervision of the whole course off illness. The commonly used antibody tests are of 4 types.
1.1Enzyme linked immunosorbent assay
Enzyme linked immunosorbent assay (ELLSA) test is the most commonly used method in primary HIV screening tests, and is applicable to the test of large quantity of samples and large scale survey. The commercial ELLSA reagents are designed to detect antibodies gP41 and P24IgG, and special ELLSA reagents can also be used to look out antibodies gP41 and P24IgM. Besides, ELLSA reagents can also distinguish HIV-1 and HIV-2 infection.
Place HIV antigens (virion, clone and complex polypeptide) at the bottom of a hole of polyethylene plate and add serum to be tested into the hole. Then through incubation, make the antibodies in the serum combine with the enzyme labeling antibodies IgG ( anti-antibodies, such as horse-anti-human antibodies) added in the serum and skim off the anti-human IGg antibodies that have not been combined. If there are HIV antibodies in the serum to be tested, the enzyme labeling antibodies will combine with the HIV antibodies, otherwise, the enzyme labeling antibodies will not be specially combined and will be skimmed away. After rinsing, add some enzyme coloration substrate to the holes. The hole that assumes a color means the serum added into it contains HIV antibodies, otherwise, the serum has no antibodies. The change of color can be measured with an auto-enzyme labeling instrument. With this method, hundreds samples can be tested at one time and the result can be obtained in several hours.
1.2 Capillary emulsion agglutination assay
Take serum 0.1ml and make it mixed with immune emulsion in a carrying groove of capillary reaction plate.
The mixed solution will flow through capillaries, where the immune emulsion will combine with the HIV antibodies of serum sample to be tested. If there are HIV antibodies in the sample, a agglutination phenomenon will appear; if no agglutination appears, the HIV antibodies are tested negative. This method is simple and quick, needing no dilution and special requirements on action temperature. Its results are easy to be judged without special instrument.
1.3 Western blot rapid assay
After infected by HIV, env encoded glycoprotein gP41 will be present in the serum of the patient. If the glycoprotein gP41 in the serum is tested positive, the patient can be diagnosed as HIV infected. The test method is: fix glycoprotein antibodies HIV-lgP41 expressed by gene engineering on a piece of nitro fibrous film and cut the film into small strips. Then hatch the serum and the strip together, and if glycoprotein antibody gP41 is present in the serum, it will be bound onto the strip. Rinse off the antibodies and serum that have not bound and put drops of golden staphylococcus albumen A(SPA) marked with horse radish peroxidase (HRPO). After secondary HRPO-SPA incubation, let the hatched substance combine with the gP41IgP of antigen-antibody complex, skim off uncombined HRPO-SPA and add color. the place where antigens are fixed on the strip combined with antibodies will assume brown, and if the antibodies are negative, there will be no color. This method is simple in operation, quick in obtaining result and needs no special instrument, and the result can be observed with eyes. Therefore, it is one of the commonly used screening tests.
1.4 Western blot assay
The principle of western blot assay is to show the specific HIV antibody proteins of the serum to be tested. It is practiced as follows: break the HIV into different protein components and use SDS-polyacrylamide gel and electrophoresis to separate out the components by their molecular weight and arrange them on the gel at different bands, forming several specific HIV protein areas. Then by adopting blot technology, transfer the proteins onto a nitro fibrous film, cut the film into test strips, on which serum added and specific protein antibodies of HIV hatched and make them combine with corresponding antigen proteins. Rinse off the non-specifically combined serum component, add enzyme labeling antibody IgG and jointly hatch them to make the enzyme labeling antibodies combine with the bound serum to be tested. After rinsing, add some color, and different specific antibodies will form different color deposition bands in different specific HIV protein areas of the nitro fibrous film. The color reaction of reaction positions can be observed by eyes, and accordingly the test results can be judged. Because different antigen proteins have different meanings in diagnosis, the appearance of different bands and types can provide accurate information for diagnosis.
With blot test, the antibodies of core protein P24 and gP41 can be detected within 2-6 weeks of primary HIV infection.
The HIV antibodies detected in serum can be used as a judgment for HIV infection. The most commonly used screening tests are enzyme linked immunosorbent assay, capillary emulsion agglutination assay and western blot rapid assay, which should be of high sensibility, with no false negative permitted, but a few false positive allowed. If the initial test results are positive, the results are confirmed using the Western blot assay.
2. How to conduct HIV antigen test?
In the course of HIV infection, not all the antigens can be detected, and antigen test is not the first choice method in the test of HIV infection. This is because in the application to the outlook of infection, the positive result always means the end of asymptomatic period and foretell the beginning of AIDS, P24 antigens can appear at the early stage of infection, and at that time antibodies can not be detected. At the late stage of AIDS and along with the lowering of P24 antibodies, P24 antigens often reappear, and this indicates the disease has already developed into AIDS and other related diseases. For about 70% AIDS patients with antigen positive, the antigen level can be constrained after taking anti-virus medicines, such as Azidothymidine，Zidovudine. Therefore, P24 antigen level is valuable in evaluating the effect of short-termntigen viruses.
Another important meaning of P24 antigen test is in the judgment of HIV infection of babies under 15months. In this period, antibodies transmitted from mother through placenta may exist in babies, and HIV infection can be confirmed not by antibody positive but by P24 antigen positive.
The time of P24 appearance is shown in the following figure:

Time of antigen appearance

Commercial reagent boxes are available for the test of HIV core protein P24 in the serum, plasma and cerebrospinal fluid. The P24 antigen test can be conducted by applying improved ELLSA, binding P24 antibodies on to the bottom small holes of polyethylene plate, enabling them capture P24 antigens in the sample, adding enzyme labeling antibody P24 antibodies, adding enzyme substrate and colored, and finally measured with enzyme labeling instrument.
Through testing P24 antigens of blood donors at transfusion center, HIV carriers who have been infected with HIV, but no antibodies having been detected and are at window stage can be found. Studies have shown that only a few of blood donors at window stage before appearance of HIV antibodies can be found. In the area where HIV spreads quickly, HIV carriers at window stage may be increased. P24 antigen test can discover HIV carriers who have been infected but can not be detected with P24 antibody test at early stage.
3.How to separate HIV? The most important evidence to confirm HIV infection is the separation of HIV from the body of patients. Viral separation needs perfect technology, complicate equipment and is expensive and time consuming, and therefore not considered as a regular diagnosis method. HIV was separated from the body of patients complicated with lymphadenopathy in 1983. Now, with improved technology, it can be separated from peripheral blood monocytes (PBMCS), serum free cells, bone marrow cells and bone marrow fluid. HIV can be cultured at the primary infection stage and symptomatic stage, but difficult at asymptomatic infection stage.
HIV separation needs high accurate tools, expensive equipment and clean laboratory to culture viruses. Virus separation is the most valuable and reliable method to evaluate HIV infection of babies because babies can get antibodies from their mothers, for which, P24 antigen test is reliable but not sensible.
The viruses separated from HIV infected can be stored in liquid nitrogen (or under a temperature of -70℃). During the storage, molecule analysis and gene analysis can be conducted and viral mutation monitored on the viruses. Recent studies have shown that in the culture of MT-2., the growing features of HIV are dentical to the disease development and antibody virus treatment reaction. In the culture of HIV, a large number of viruses are generated from non- multinuclear shape mutation, and this is closely related to the rapid development to AIDS and not sensible to azidothymidine，zidovudine.
How to conduct HIV nucleic acid test?
Some viruses can be detected with nucleic acid crossbreed technology. This technology involves the adhesion of label nucleic acid probes, the isogenous matching of viral chromosome sequence and the location of viral DNA on filter paper, or gel or HIV-infected cells (hybridization in situ). The adhesion of label nucleic acid probes can be confirmed with radioautography or color reaction inducted by enzyme links. In situ hybridization technology can also be used to study HIV infection course, especially fixed tissue test. HIV nucleic acid test is not sensible because HIV nucleic acid is present only in a small portion of cells in peripheral blood. In different patients and at different time of serum antibody converting to positive, the ratio of peripheral blood lymphocytes that contain HIV nucleic acid is different, usually between 0.01-0.1percent.
At present, polymerase chain reaction (PCR) is usually used to test HIV nucleic acid. This method is a new technology that can increase dramatically the specific DNA of HIV in a short time. By application of this method, trace pathogenic genes can be detected, so that it is an accurate and reliable HIV nucleic acid test method for the diagnosis of AIDS in terms of its strong sensibility and high specificity, especially in the detection of viruses in the peripheral blood cells of HIV infected patients at asymptomatic stage, which are few in number and difficult to detect with regular method. But with PCR method, a higher positive reaction can be obtained, thus, the position of nucleic acid test in the diagnosis of AIDS and HIV infection has been greatly improved. However, because of its high sensibility, PCR method has also false positive in test of slightly contaminated samples.