TRI-DOT Test for HIV Detection - All About a Rapid Screening Assay

Introduction

Human immunodeficiency virus (HIV) causes acquired immunodeficiency syndrome (AIDS), which has been considered a global epidemic in recent decades. AIDS is a significant threat to public health as it causes the complete failure of human immune mechanisms and makes them susceptible to fatal opportunistic infections. Also, no specific cure exists for the illness, which further implies the importance of AIDS prevention and early HIV detection.

Diagnosing HIV infection becomes inevitable for preventing and managing infected individuals. Several HIV diagnostic assays have been developed through research and clinical studies. In the current scenario, there is an increased demand for rapid HIV screening assays as they are simple tests that favor accurate and early detection. The HIV TRI-DOT test is one such rapid screen immunoassay specifically developed for detecting HIV antibodies, and this article discusses its various clinical dimensions in detail.

When Is the HIV TRI-DOT Test Performed?

According to CDC (the Centers for Disease Control and Prevention), it is scientifically proven that no tests are available to detect HIV in the blood immediately after viral exposure or suspected infection. This is because of its peculiar window period, which commonly refers to the time lapse between the point of infectious exposure and the progression of detectable specific antibodies in the suspected individual’s serum or plasma. The CDC has stated that the window period for HIV antibody detection ranges from 18 to 45 days post-exposure.

The indications for performing the HIV TRI-DOT test are as follows-

• The HIV TRI-DOT test is indicated for rapid screening from 23 to 90 days following infectious exposure.

• It is widely used in hospital settings where emergency surgeries or high-risk surgical interventions are to be performed without delay.

• It is predominantly used in screening pregnant women and obstetric delivery rooms to prevent HIV transmission from mother to baby.

• It is also used for HIV screening in neonates to rule out maternal-fetal HIV transmission.

• The HIV TRI-DOT test is the preferred screening method for HIV testing in voluntary counseling and testing (VCT) centers, where tests are conducted with proper pre-test and post-test counseling as per WHO (the World Health Organization) guidelines.

• It is extensively helpful for HIV screening in blood banks and peripheral hospitals.

• This test is performed in healthcare facilities with limited resources and minimal laboratory expertise.

What Are the Uses of the HIV TRI-DOT Test?

The HIV TRI-DOT test is a screening test performed to detect HIV antibodies in human plasma and serum. It is a qualitative assay that indicates the presence or absence of HIV infection in a particular individual.

The salient features of the HIV TRI-DOT test are as follows-

• It is a rapid, accurate, visual, and sensitive immunoassay used to detect both HIV-1 and HIV-2 antibodies.

• It is a screening test used only for in-vitro diagnostic purposes.

• Clinical studies have proved that the HIV TRI-DOT test is more suitable than ELISA (enzyme-linked immunosorbent assay), especially in limited healthcare settings.

• Unlike ELISA, the HIV TRI-DOT test is cost-effective and could be used to perform tests on small batches of samples.

• The accuracy of the HIV TRI-DOT test is 99 percent, with a specificity of 99.9 percent and a sensitivity of 99.5 percent.

• It is easy to use, requires minimal technical training, is visually interpreted, and the reagents have a longer shelf-life.

What Is the Principle of the HIV TRI-DOT Test?

Two forms of HIV exist, namely, HIV-1 and HIV-2, and both are capable of causing infections in humans. These viruses are classified as slow viruses and belong to the retrovirus family. They are enveloped viruses, and the proteins in their envelope are antigenic (induce an immune response). The working principle of the HIV TRI-DOT test is based on eliciting these antigen-antibody reactions in a visual field.

The HIV TRI-DOT test works in the following ways-

• When HIV enters the human body, the first immune response is the appearance of the p24 antigen in the individual’s serum. However, this antigen is short-lived and is overtaken by anti-HIV antibodies (anti-HIV-1 and anti-HIV-2) due to seroconversion (the first appearance of antibodies in blood to any infection).

• HIV-1 and HIV-2 antibodies produced are predominantly IgM, IgG, and IgA immunoglobulin (Ig) types. The HIV TRI-DOT is designed to detect all types of antibodies (immunoglobulin types) if they are present in human plasma or serum (blood samples).

• HIV antigens (immunodominant regions in HIV-1 and HIV-2 envelopes) are immobilized, using recombinant DNA technology, on an immunofiltration porous membrane.

• When samples and suitable reagents are allowed to pass through this immunofiltration absorbent membrane, HIV antibodies (if present in the test sample) get captured by these immobilized antigens.

• This step adds protein-A conjugate (a visualization agent) to the membrane. This conjugate binds with the complement (Fc portion) region of the captured HIV antibodies, giving a distinctive pinkish-purple dot or dots against the white background, indicating the presence of HIV antibodies in the test samples.

How Is the HIV TRI-DOT Test Performed?

The HIV TRI-DOT test is a kit-based rapid screening method where all the reagents are in ready-to-use forms. The kit contains micro-welled devices, each containing in-built three antigen-coated dots (a quality control dot, an HIV-1 dot, HIV-2 dot). The HIV TRI-DOT test involves handling biohazardous and infectious components, so the test should only be performed by a healthcare provider in sterile clinical settings.

The following procedures are precisely executed to achieve maximum specificity and prevent errors.

• Sample Collection and Processing - Human blood samples are collected in dry, sterile vials and centrifuged to separate the serum or plasma at room temperature. Though fresh samples are preferred, samples could be stored at two to eight degrees Celsius (35.6 to 46.4 degrees Fahrenheit) if the assays are not conducted immediately. The serum samples could even be frozen and stored at -20 degree Celsius (-4 degrees Fahrenheit). However, before starting the assay, the preserved samples must be thawed (allowed to reach room temperature). Samples contaminated with blood, microbes, or other particulate matter cannot be assayed and are discarded following biosafety protocols.

• Handling Reagents - The HIV TRI-DOT test kit contains buffer solution and protein A conjugate as ready-to-use reagents in separate containers with closed-sealed nozzles and screw cap lids. The opening of reagents and the procedural sequence of adding them should be carried out in a sterile environment to prevent external contamination.

• Test Procedure - Three drops of buffer solution are added to the device at its center. Then, 50 µl (microliter) of test plasma or serum is added using a separate sample dropper, followed by subsequent step-wise additions of buffer (5 drops) and protein-A conjugate. Another five drops of buffer solution are added before reading the results.

• This test is a rapid method, with results obtained within three minutes. However, allowing the reagents to get absorbed completely inside the device is essential before adding consequent solutions.

• Precautions - All samples are potentially considered hazardous and capable of transmitting infections. Using disposable gloves and optimal biohazardous suits is mandatory while performing the entire HIV TRI-DOT, from sample collection to disposal of used devices. Strict biosafety disposal protocols must be followed while discarding tested TRI-DOT devices and unused samples.

How to Interpret the HIV TRI-DOT Test?

Results of the HIV TRI-DOT test should be interpreted with a wide understanding of the window period and progression of HIV infection.

• If the control dot turns into a pinkish-purple dot, it indicates the device and the reagents are working. If only the control dot develops color, the test sample does not contain HIV antibodies and is reported as non-reactive.

• If the control and HIV-1 dots (two dots) turn pinkish-purple, it is reported that the sample is reactive to HIV-1 antibodies.

• Likewise, if the control and HIV-2 dots (two dots alone) change color, it indicates HIV-2 antibody reactive.

• If the control and HIV-1 and HIV-2 dots (all three dots) turn pinkish-purple, the test sample is reactive to HIV-1 and HIV-2 antibodies.

• The HIV TRI-DOT test is only a rapid screening method, and reactive test samples should be confirmed through ELISA and HIV western blot tests.

• According to WHO and UNAIDS (the Joint United Nations Programme on HIV/AIDS) recommendations, approved HIV testing algorithms should be followed for confirmatory diagnosis of HIV infection or AIDS.

Conclusion

HIV testing is extensively carried out to detect and prevent the spread of HIV infection. Rapid tests like the HIV TRI-DOT test are extremely helpful in screening HIV antibodies at the current HIV prevalence. Though not a confirmatory test, The HIV TRI-DOT method is more accurate and beneficial for HIV screening in limited-resource healthcare facilities like peripheral hospitals, VCT centers, and blood banks. Early detection of HIV antibodies not only helps in prevention but also prompts the initiation of antiretroviral therapy (ART), thereby ensuring the general wellness of the affected individuals is efficiently maintained.