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Can we use the HIV antibody test to detect HIV in newborns?

Can we use the HIV antibody test to detect HIV in newborns?



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Adults and elder children can use HIV rapid test to test HIV infection. Can we use the HIV antibody test to detect HIV in newborns too?


The rapid screen usually tests the oral mucosa by swabing the inside of the cheek and checking for the presence of anti-HIV envelope antibodies. Although the precise target of these antibodies seems to be kept propriatary, my guess would be the detection of either p24 antibodies or envelope antibodies that bind the V3 region.

Regardless, the point is they test for the presence of antibodies, which a child does not start making until they are born. See this post for a great explanation

Since a child has mostly maternal antibodies, the presence of anti-HIV antibodies would only mean that the mother has started producing antibodies to HIV (Seroconversion). This means that although the child has been exposed, they may themselves not be infected.

The mucosa, particularly in the oral region has a very low titer of anti-HIV antibodies, so I think this test would probably be best to direct the physician to conduct additional tests that are more indicative of HIV infection like p24 western blot.


Comprehensive, up-to-date information on HIV/AIDS treatment and prevention from the University of California San Francisco

Although HIV antibody tests are the most appropriate for identifying infection, alternate technologies can contribute to an accurate diagnosis, assist in monitoring the response to therapy, and can be used to effectively predict disease outcome. Viral isolation through viral culture, nucleic acid tests to detect viral RNA, and tests to detect p24 antigen can be used to demonstrate virus or viral components in blood, thereby verifying infection. These methods are highly specific, and a positive result confirms infection. Each has limitations, however, and their use must be tailored to proper testing situations. Tests for viral nucleic acid have recently been introduced, but require sophisticated technology and dedicated, well-trained personnel.

The HIV antigen test is currently used for screening blood for transfusion and is appropriate for use in several other testing situations. It offers the advantages of simplicity and cost effectiveness for verifying infection, but is less than perfect.

The p24 antigen assay measures the viral capsid (core) p24 protein in blood that is detectable earlier than HIV antibody during acute infection. It occurs early after infection due tothe initial burst of virus replication and is associated with high levels of viremia during which the individual is highly infectious.(1) When antibodies to HIV become detectable, however, p24 antigen is often no longer demonstrable, most likely due to antigen-antibody complexing in the blood. When detected, p24 antigen is highly specific for infection. The specificity of the p24 antigen test in detecting HIV infection using PCR as the gold standard was calculated from data using test kits from 2 manufacturers to be 99.9%.(2) In the same study, the sensitivity following a procedure of neutralization to confirm the presence of p24 antigen (see below) was 100%.

Testing for p24 can be of value in: (1) detecting early HIV infection, (2) screening blood, (3) diagnosing infection in the newborn,(3) and (4) monitoring antiviral therapy.(4) A major limitation is that the test is insensitive when testing blood, both because low levels of antigen are difficult to detect, and because antigenemia occurs only transiently during different stages of infection.(5) In fact, the HIV antigen test is incapable of detecting 75% of blood donors who are infected but seronegative.(6)

p24 antigen is found in serum in either free form or bound by anti-p24 antibody. Free p24 can be measured with enzyme immunoassays whereas detection of bound p24 requires pretreatment with an acid to dissociate the complex. Procedures to dissociate antigen-antibody complexes have improved the sensitivity of the p24 test, but antigen remains undetectable in most asymptomaticpatients.(7) Antibody remains detectable throughout infection, whereas p24 antigen characteristically appears early and late during infection.

Following HIV infection, the sequence of markers to identify infection in their chronologic order of appearance in serum are: viral RNA, p24 antigen, and anti-HIV antibody. About 2 weeks after infection, viremia is thought to increase exponentially and then decline to a steady-state level as the humoral and cell-mediated immune responses control HIV replication. This time interval, the serologic "window period," is characterized by seronegativity, occasionally detectable antigenemia, viremia (as measured by RNA), and variable CD4 lymphocyte levels. Detection of specific antibody to HIV signals the end of the window period and labels the individual as seropositive.

The exact time when HIV RNA, antigen, and antibody can be detected depends on several factors, including the test used, individual host responses, and viral characteristics. Viral RNA can be detected within the first 2 weeks using the highly sensitive RT-PCR method. Antigen, although transient, can appear as early as 2 weeks after infection and lasts 3 to 5 months.(8) Newer generation antibody assays, including the HIV third generation antigen-sandwich antibody assays, can detect antibody in most individuals at about 3 to 4 weeks post-infection.(9) Latein the course of infection, i.e., as progression to AIDS occurs, anti-p24 antibody decreases and p24 antigen again becomes detectable.(10) The decline in antibody to p24, the production of antigen, and the generation of immune complexes are most likely related to changes in viral replication,(11) because antibodies to other components (e.g., envelope glycoproteins) persist throughout infection.(12)

In summary, for the screening of blood or for diagnosis, the current serologic window period represents a period of about 3 to 4 weeks when HIV infection cannot be demonstrated using antibody tests. Methods that detect p24 antigen can shorten this period by about 1 week, although not all seronegative HIV-infected individuals will be identified. Tests that detect viral RNA can reduce this window period by another several days over antibody and antigen identification, but these assays are not currently suitable for routine laboratory use due to their requirement for sophisticated equipment and technical expertise. Therefore, the period after infection when diagnosis is possible may be reduced to 2 weeks or less if an all-inclusive testing strategy utilizing antibody, p24 antigen, and RNA detection methods is employed.

There are several uses, and mis-uses, of the p24 antigen assay. It is important to realize that the p24 antigen test detects soluble p24 antigen, presumably following viral replication, and does not specifically identify live virus. Therefore, a positive antigen test does not confirm that a sample is infectious, and should not be used for that purpose. The only means available to demonstrate that a sample contains infectious virus is by virus isolation and culture. A negative result for antigen does not rule out infection, because the test lacks exquisite sensitivity i.e., the test should not be used to verify noninfection. Antigen detection signals infection, however, and positive results in seronegative individuals can be an effective, although not cost effective, means to identify early infection.

The p24 antigen test is most useful in detecting antigen in supernatants from cultures that have been inoculated with cells from a patient suspected of being infected (viral culture and isolation). The antigen test is the method of choice for detecting the presence of free antigen in culture, and it is two orders of magnitude more sensitive than the reverse transcriptase assay.(13) The test is most useful for detecting viral antigen in culture fluids because large quantities of antigen are exuded in the supernatant fluid during viral replication.

The antigen test can be performed on fluids other than those of culture and serum. Evidence determined from the testing of cerebrospinal fluid (CSF) indicates that many patients with HIV dementia and encephalopathy have detectable antigen in CSF, most likely due to active replication of the virus in cerebral tissue.

The p24 antigen test can be of value in blood screening, for identification of acute infection, for monitoring infection, andto assist in the diagnosis of infection in the newborn (each is discussed subsequently). It has been used for detecting early infection in rape cases, identification of infection after occupational exposure, and for assisting in the resolution of indeterminate Western blot results. The degree to which p24 antigen assays can detect p24 antigen from all clades of HIV-1, HIV-2, and HIV-1 Group O, however, is unknown.

In the United States, transmission of HIV by blood transfusion occurs almost exclusively during acute infection when the donor is seronegative. In 1995, antigen testing was instituted to supplement antibody screening of donated blood. Prior to this date, it was estimated that 1 donation in every 210,000 to 1,140,000 was made by an HIV-infected individual during the window period, which is usually 22 to 25 days, but may be longer.(9) On the basis of this assessment of risk , there could be 18 to 27 units (32 to 49 infectious components) of HIV-infected seronegative blood donated per year.(9) By instituting p24 antigen screening of blood, an estimated 4 to 6 cases of transfusion-associated HIV infections can be prevented per year, lowering the estimated risk per unit transfused to a range of 1:562,000 to 1:825,000.(9,14) The risk varies in the United States according to geographic regions, however, ranging from zero in low incidence areas to 1 in 70,000 in high incidence areas.(9) By late 1996, 4 cases of HIV-1 transmission by HIV-1 seronegativeblood donors were reported during that year.

With the goal of attaining the safest blood supply in the world, the p24 antigen assay was recommended by the U.S. FDA for use in the screening of blood, blood components, source leukocytes, and source plasma targeted for transfusion.(15) To achieve this goal, the p24 antigen test must be performed in addition to testing for HIV antibody. Donors who test positive for p24 antigen (confirmed) are permanently deferred from donation. Donors who test indeterminate for p24 antigen (see below) should be temporarily deferred from donation for a minimum of 8 weeks.(15) Subsequently, the donor can be reinstated if results of antigen and antibody tests are negative. Donors are permanently deferred if the screening test is repeatedly reactive on any subsequent testing.

This mandate to reduce the risk of transfusion-induced infection has generated controversy, because the cost of routine screening of blood donors for p24 antigen may be in the 100 million dollars per year range, a cost far in excess of other health interventions.(16)

Many experts agree that acute HIV infection is identifiable before seroconversion in the majority of cases, both clinically and through the use of laboratory methods.(17) The use of the p24 antigen test can assist in the diagnosis of acute infection prior to seroconversion in most infected individuals. Prompt diagnosisallows early counseling to minimize transmission by decreasing high risk behavior and aborting exposure when levels of viremia are high and infection is at a highly contagious stage. Furthermore, it permits early tracing of individual contacts, and can provide a unique opportunity to study the physiologic effects of acute infection.

Identification of early infection allows for the institution of early therapy (which is now available), thereby potentially decreasing dissemination of the virus. Many clinical laboratories are using rapid HIV antibody tests to detect infection in the source patient following exposure cases in order to institute therapy at the earliest time following exposure. In high-risk source patients who are seronegative, the p24 antigen assay may be similarly used for detecting early infection so that treatment can be instituted in a time frame that is clinically relevant.

The U.S. Public Health Service guidelines discourage the use of routine testing for p24 antigen in settings other than blood and plasma centers as a method for diagnosing HIV infection because the estimated average time from detection of p24 antigen to detection of HIV antibody is 6 days, and not all recently infected persons have detectable levels of p24 antigen.(18)

At the 1987 International AIDS Congress in Washington, D.C., and in Stockholm in 1988, it was reported that a decline in theserum titer of antibody to the p24 protein and a rise in p24 antigen occurs as an HIV-positive individual progresses to the most serious phase of the disease.(19) In the past, before the availability of viral load testing, the p24 antigen assay was used extensively for monitoring the development of AIDS and for charting disease progression. Although many clinicians are now monitoring infection using viral load testing, the p24 antigen assay continues to provide information at a much reduced cost and with faster turn around times. Recently, its use in predicting who may rapidly progress to AIDS (rapid progressors) by noting a higher prevalence of p24 antigenemia at the first seropositive visit has been reported.(20) p24 antigen testing is used less often, however, for monitoring the effectiveness of anti-HIV drug regimens now that RNA levels can be determined.

Diagnosis of HIV infection in the newborn is problematic because of the omnipresence of maternal HIV antibody during the first year of life. Testing the newborn inevitably results in the detection of maternal antibody, with both the newborn and the mother having identical antibody profiles. After approximately 12 months, maternal antibody wanes in the newborn and a diagnosis based on the newborn's antibody becomes possible. A newborn must be monitored using antibody tests for up to 18 months, however, because even after maternal antibody disappears, it may be several more months before the newborn's immune system iscompetent enough to produce antibodies. In fact, an infected newborn whose maternal antibody has disappeared at 12 months may become seropositive at 18 months. In some cases, demonstration of an increase in antibody titer over time in the newborn can suggest true infection.

Specific IgM antibody tests have not proven to be reliable.(12) Because of the uncertainty of infection status in the newborn when using antibody tests, the detection of viral nucleic acid, p24 antigen, and viral isolation are of value and can offer help in making an early diagnosis.(21) The use of either virus isolation or viral DNA detection methods has allowed a greater than 90% prediction of infection after 1 month of age,(22) and a 97% prediction by 3 months.(23) The detection of p24 antigen in newborns is less sensitive (50 to 80%) but can achieve similar sensitivities by 6 months. At birth, the combined use of p24 antigen, viral culture, and DNA PCR allows a prediction of infection of only 50%, although the specificity is 100%.(24) A negative PCR result in the first month of life confirms noninfection with a probability of greater than 90%(25). It is clear that better methods to confirm early infection in the newborn are still needed.

p24 antigen may not be readily detected (particularly in asymptomatic infections) because its concentration in serum may be low due to low viral replication. The antigen test isrelatively insensitive, being able to detect only about 10 pg/ml of p24 antigen. This quantity of antigen may not be present in the serum of infected individuals, even when the virus is actively replicating. In fact, only about 50 to 60% of AIDS patients, 30 to 40% of AIDS-related complex (ARC) patients, and 10% of asymptomatic patients will have p24 antigenemia.

Furthermore, the degree of antigenemia seems to vary depending on the population tested. For example, several studies have shown that African patients with AIDS have higher levels of antigen than a similar group of individuals in the United States.(26) Conversely, African patients infected with HIV but who are asymptomatic have been shown to have lower levels of p24 antigenemia than a similar group in the United States.

One reason for the lack of sensitivity of the antigen test when testing the serum of HIV-infected persons is that free p24 antigen in serum may be complexed with p24 antibody. The test cannot detect complexed antigens. Even with complex dissociation, however, the sensitivity remains low and the test can only detect about 50% of asymptomatic patients.(7)

Although procedures vary between manufacturers, HIV p24 antigen tests employ ELISA technology with modifications to detect antigen, not antibody. In a representative assay, such as an "antibody sandwich" type, a specific monoclonal antibody toHIV p24 is attached to the solid phase (microtiter plate-well or polystyrene bead) acting to "capture" the viral antigen in the sample when added. The sample is diluted in a Triton X100 detergent to disrupt virions, and if antigen is present in the serum, the antigen will attach to the monoclonal antibody on the solid phase. Following a wash step, an antibody detector is added and incubated. This detector reagent is usually a high-titer antibody to p24 antigen that is coupled to biotin. Subsequently, incubation with a conjugate (streptavidin-peroxidase) labels the complex by attaching via biotin. An avidin-biotin system acts as an amplifier to generate additional signal to detect the small quantities of antigen in the sample. Addition of a substrate (tetramethylbenzidine) will allow the production of color as the enzyme cleaves the substrate. A weak acid (e.g., 2 M sulfuric) is finally added to stop the reaction after a defined period of time. Resultant optical density values are proportional to the amount of HIV-1 p24 antigen in the specimen. This assay can detect p24 antigen in the pg/ml-to-ng/ml range. The optical density is read with a spectrophotometer at 450 nm.

The p24 antigen tests are subject to false-positive reactions, presumably due to interfering substances and immune complexes. Therefore, specimens that test reactive in the antigen test must be confirmed, using a more specific method. Anantigen neutralization assay is used to verify specificity. This neutralization assay is purchased as supplemental reagents for the p24 antigen assay. The supplement includes a neutralization reagent used in a pretreatment step before repeating the p24 antigen test.

The sample, presumably containing the antigen, is incubated with a neutralizing reagent that is a human anti-p24 antibody. During this incubation, if antigen is present, it will be complexed with the neutralizing antibody and prevent p24 antigen from being bound by the solid-phase capture reagent in the p24 antigen assay. Subsequently, the antigen assay is repeated on this preincubated sample along with an aliquot of the same sample that has not been preincubated with the neutralization reagent the O.D. readings are compared. For the sample to be considered confirmed positive for antigen, the O.D. readings of the sample following the neutralization must be reduced by at least 50% compared to the O.D. readings of the nonneutralized aliquot. If this degree of reduction (inhibition) does not occur, the sample is not confirmed for antigen, and the reactivity was probably not due to p24 antigen further resolution is necessary by RNA testing or follow-up testing.

Because all assays are performed in duplicate or quadruplicate, nearly 1 ml of serum is required to complete the test for a final confirmed result. The test is also relatively expensive, at least twice as expensive as the antibody tests. In addition, depending on the number of replicates needed and thetotal number of samples being tested, costs can become exorbitant. A protocol for an in-house p24 antigen assay designed for testing large numbers has been described and is more cost effective than commercially available assays.(27)

To determine the levels of p24 antigen in blood, an HIV-1 antigen standard is diluted to prepare a series of six standards of varying concentrations. Concentrations vary between 0.0 and 125 pg/ml. A standard curve is generated from which optical density values of the unknown specimens are interpolated to determine their concentration. The standard curve is constructed using a linear graph and plotting the concentration of the HIV-p24 antigen standard (pg/ml) on the X-axis versus the mean optical densities for each standard on the Y-axis. Each standard is added in duplicate wells, and at least 5 controls must be included (3 negatives and 2 positives). If the value of the unknown sample is higher than the value of the highest standard, the sample must be diluted in normal human serum and the entire neutralization procedure is repeated.

To improve sensitivity of the p24 antigen assay, manufacturers introduced an Immune Complex Dissociation (ICD) procedure using low pH to dissociate p24 antigen/anti-p24antibody complexes before performing the antigen assay. Using this procedure, an increased sensitivity of the assay was demonstrated, particularly for asymptomatic HIV-infected individuals. This dissociation procedure allows for detection of both free p24 antigen and complexed p24 antigen/antibody. The method not only increases the number of antigen positive individuals (epidemiologic sensitivity), but also can detect lower amounts of p24 antigen (analytical sensitivity).(28) Although ICD may result in an overall increase in sensitivity of the antigen test, detection of all HIV-infected individuals is only about 50%.

More recently, a report has indicated that the p24 antigen assay can be modified to increase its sensitivity for detecting early HIV infection to a level approaching that of the PCR (viral load) test, and may be more effective in monitoring disease and predicting outcome.(29) They describe a modification to the immune-complex dissociation p24 antigen assay procedure in which a signal-amplification step is used as a final step. This amplification involves the addition of a tyramide compound which generates an intermediate to produce more enzyme and substrate molecules and hence, more signal. This allows smaller quantities of p24 antigen to be detected. Since the p24 antigen assay by itself has never been very sensitive (unless when used for testing culture supernatants), this modification could prove to be very useful. Its advantages over PCR include its simplicity, cost effectiveness, and high throughput capability. Further investigations will need to be performed to verify its performance characteristics and usefulness.

Upon initial testing by the p24 antigen test, the results are labeled as reactive or nonreactive based on the calculated cutoff value. If reactive, the test is repeated in duplicate using the same sample source. If the initial result is nonreactive, or if the initial result is reactive but both replicates on repeat testing are nonreactive, the sample is labeled as negative. If at least 2 results are reactive (repeatedly reactive), the sample is presumptive for containing p24 antigen, and must be confirmed using the neutralization assay.

Following testing by the p24 antigen neutralization test on samples that are repeatedly reactive, the results can be classified as positive, or indeterminate (either non-neutralizable or not meeting the criteria for acceptability). Repeatedly reactive p24 antigen tests that are not positive in the neutralization test are unlikely to be from infected individuals. Those sera that meet the criteria for positive by neutralization are considered to be from truly HIV-infected individuals.

Several criteria must be met to validate the neutralization (confirmation) test: (1) the optical density reading of the sample that has received a negative (non-neutralizing) reagent must be greater than the sum of the mean value of normal human serum plus a constant, (2) the optical density reading of the aliquot that received the neutralization reagent must be decreased by at least 50%, and (3) each control must produce optical density reading within the ranges stated by the manufacturer. The test materials include wells for background controls that are used to subtract values caused by nonspecific reactions. Neutralizibility of the sample containing the neutralizing reagent is calculated using the following formula:

To validate that the quantitative p24 antigen test has performed as expected, the optical density reading of the standard must meet certain criteria. These criteria vary according to manufacturer, but usually require that the mean optical density of the 0 pg/ml standard and the substrate blank must be less than 0.100, and the mean optical density value of the 62.5 pg/ml standard must be greater than or equal to 0.500.

Most HIV-infected individuals produce antibodies to p24 in concentrations that vary from bleed to bleed, and hence affect the ability to detect and quantify p24 antigen. Even after immune complex dissociation, host antibodies may recombine with p24 antigen, affecting p24 antigen detection by competing with the capture reagent (solid-phase monoclonal antibody). The rate of recombination depends on the concentration of p24 antigen and anti-p24 antibodies, the affinity of the antibodies, and the time and temperature of the reactions.

Because of the potential for rapid degradation of HIV-1 p24 antigen in improperly stored samples, individuals who have indeterminate neutralization results should be retested using a new specimen. The most common causes of an indeterminate neutralization test (when criteria are not met) are: nonspecific reaction in an uninfected person, low p24 antigen levels in an infected person, or sample deterioration or antigen-antibody complex formation during storage.(18)

The Public Health Practice Program Office of the CDC's Division of Laboratory Systems has implemented a Laboratory Performance Evaluation (PE) Program to assess performance for p24 antigen testing.(30) Participants receive assessment panels of sera twice a year and report results to the CDC. The CDC then prepares and distributes reports to the participating laboratories describing the suitability of their testing practices. The program is voluntary and free of charge (telephone number 770-488-4366).

It is advisable to include an external control when performing the p24 antigen assay. Such a control, different from the internal controls included in the test kit, provides a means to validate the entire procedure (including the ability of the internal controls to produce appropriate cutoff values), and to monitor intra-run, inter-run, and test kit lot-to-lot variations. A detailed description of the use of external controls for this purpose has been published.(12) A variety of standards for p24 antigen testing are available.(31)


Preventing Perinatal HIV Transmission

Advances in HIV research, prevention, and treatment have made it possible for many women with HIV to give birth to babies who are free of HIV. The annual number of HIV infections through perinatal transmission in the United States and dependent areas a has declined by more than 95% since the early 1990s.

*Viral suppression is defined as having less than 200 copies of HIV per milliliter of blood. An undetectable viral load means having a viral load so low that a test can&rsquot detect it. The benefits of having an undetectable viral load also apply to people who stay virally suppressed.

All women who are pregnant or trying to get pregnant should encourage their partners to also get tested for HIV. If either partner has HIV, that partner should take HIV medicine daily as prescribed to stay healthy and prevent transmission.

Taking HIV medicine every day can make the viral load undetectable. People who get and keep an undetectable viral load (or stay virally suppressed) have effectively no risk of transmitting HIV to HIV-negative sex partners.

For babies with HIV, starting treatment early is important because the disease can progress quickly in children. Providing HIV medicine early can help children with HIV live longer, healthier lives.


HIV Testing

The federal Centers for Disease Control and Prevention (CDC) estimates that 38% to 44% of all adults in the United States have been tested for HIV and that between 16 and 22 million people aged 18-64 years are tested for HIV annually. However, of more than one million people living with HIV in the U.S., it is estimated that about one-quarter (252,000 to 312,000 persons) are unaware of their HIV status. Because they do not know they are infected, these individuals are unable to benefit from clinical care related to their HIV disease. In addition, some of these people may unknowingly be transmitting HIV to other people. The San Francisco AIDS Foundation strongly encourages people who are at risk for HIV to be tested regularly. Whether or not to take the HIV antibody test, however, is a personal decision that only you can make for yourself. That being said, here are some things you may wish to consider:

  • Knowledge is power.
  • If you find out that you are HIV-positive (infected with HIV), you and your healthcare providers can better plan early treatment and intervention, improving your chances of slowing down the progression of HIV disease.
  • If you test negative, you may feel less anxious.
  • By knowing your status, you can find out whether or not you can infect others, and what precautions you might take to prevent transmission to other people.
  • Regardless of the result, testing tends to increase your commitment to overall good health habits. If you test positive, you can learn more about HIV and be proactive in taking care of your health.
  • If you are considering having a baby, you can take advantage of treatments that potentially prevent transmission of HIV to the baby.

What Are HIV Antibody Tests?

As the body fights viruses, the immune system creates antibodies to that virus. HIV antibody tests do not measure or detect the virus itself but instead look for your body's reaction to the virus -- the presence of antibodies to HIV.

ELISA

The ELISA (also sometimes called EIA) is often used as the first screening tool. It is inexpensive and very sensitive for detecting the presence of HIV antibodies. In most cases, a blood sample is tested, but other types of ELISAs that use saliva and urine have also been developed. The actual ELISA takes 3.5 to 4 hours, but most test sites send samples to outside labs, where they are tested in batches, so you may have to wait one to two weeks for results.

Beyond the Window Period ELISA tests are very rarely "false negative." This means if you have a negative test result, and you had met the Window Period guidelines after the last potential exposure, you are really HIV negative. An ELISA test may rarely be "false positive." False positive ELISA results can occur if someone is tested right after events that temporarily stimulate the immune system, such as viral infections or immunizations. They could also occur because of lab error, or because of the test's very high sensitivity, discussed below.

For these reasons, positive ELISA results must always be confirmed with a Western Blot or IFA (below), and at reputable test sites this is commonly done automatically -- meaning you don't have to have another blood sample drawn.

A relatively new test, called a detuned ELISA, which has been used in research settings, will soon become more widely available to other test sites. The detuned test, which is used only after HIV antibodies are confirmed by a Western Blot test, can determine if the HIV infection is recent (within the last six months), which may be useful for deciding upon possible early treatment options.

Western Blot (WB) Assay

The WB is a confirmatory test: it is only performed if an ELISA or rapid test is positive. The WB can be positive, negative, or indeterminate. Indeterminate tests are neither positive nor negative. An indeterminate result usually means that a person has just begun to seroconvert at the time of their test. In the rare cases in which this occurs, the person will need to be retested, usually about one month later. False positive results are extremely rare with the WB, so it confirms (proves) that HIV antibodies are present.

Indirect Immunofluorescence Assay (IFA)

The IFA can be used instead of the WB to confirm ELISA results. Like the WB, IFA tests for the presence of antibodies in a blood sample. The exact strategy is slightly different in that it uses a microscope. It can be faster than a WB, so the few labs that use it can get results to the patient more quickly.

Rapid Tests for HIV

Rapid testing for HIV has become one of the most prominent ways that people receive HIV tests today. In as little as 20 minutes, you can find out your HIV status.

Rapid tests work similarly to traditional HIV test: they look for antibodies to HIV, not the virus itself. Most commonly, a rapid test is adminestered by sampling the oral mucosa (the mucous that is everywhere in your mouth) and putting it through a test to see if HIV antibodies are present.

Some people may think that, since the test uses the mouth to test for HIV, that HIV can be transmitted to other people through saliva, spit and/or oral mucosa via kissing, sharing utensils, or sharing glasses. This is not true. For more information, see this website's section on How HIV is Spread.

A rapid test runs for 20-40 minutes, after which you will receive a result. Rapid tests are extremely accurate. However, since rapid tests have a small margin of error (ranging from 1 to 3 per 1,000), all positive (reactive) results have to be put through a confirmatory test.

The most common type of rapid test currently in use in Callifornia is the OraQuick Advance. The availability of rapid tests varies by city. Some testing locations in California charge for this type of test, but there are many locations that will provide the test for free. For testing locations, you can call the California HIV/AIDS Hotline at 1-800-367-AIDS or you can do your own referral search by visiting www.aidshotline.org.

Window Period

The "window period" is the time it takes for a person who has been infected with HIV to react to the virus by creating HIV antibodies. This is called seroconversion.

During the window period, people infected with HIV have no antibodies in their blood that can be detected by an HIV test, even though the person may already have high levels of HIV in their blood, sexual fluids, or breast milk.

Here is what the CDC says about the window period:

"Antibodies generally appear within three months after infection with HIV, but may take up to six months in some persons."

This CDC definition of a three to six month window period has been commonly used for a number of years.

What does this mean for you?

The three month window period is normal for most of the population. Many people will have detectable antibodies in three or four weeks. Very, very rarely (i.e., only a few cases ever), a person could take six months to produce antibodies.

You may be anxious to be tested soon after an encounter which you perceive to be risky (for a discussion of what behaviors put you at risk for HIV and which ones do not, see the section on How HIV Is Spread). You want to know: can I be antibody tested without waiting three months? How accurate is the test after, say, six weeks?

Unfortunately, we simply don't know.

Think about this: if you got a negative test at six weeks, would you believe it? Would it make you less anxious? If so, then go for it. But to be certain, you will need to be tested again at three months. Some test centers may recommend testing again at six months, just to be extra sure.

Although HIV may not be detected by a test during the window period, HIV can be transmitted during that time. In fact, individuals are often most infectious during this time (shortly after they have been exposed to HIV).

Interpretation of Test Results

A positive (reactive) result means:

  • You are HIV-positive (carrying the virus that causes AIDS).
  • You can infect others who come into contact with your blood, semen or vaginal fluid. You should take necessary precautions to avoid transmitting HIV to others.

A positive result does NOT mean:

  • You have AIDS.
  • You will necessarily get AIDS.
  • You are immune to AIDS, even though you have antibodies.

A negative (non-reactive) result means:

A negative result does NOT mean:

  • You are not infected with HIV (you may still be in the "window period").
  • You are immune to HIV.
  • You have a "resistance" to infection.
  • You will never get HIV.

An indeterminate result (which is rare) means:

  • The Western Blot (WB) result is unclear. The entire HIV test must be repeated with a new blood sample, usually several weeks after the first blood test.
  • Indeterminate results usually occur if the test is performed just as the person begins to seroconvert.

Accuracy of Antibody Tests

Antibody tests are extremely accurate, whether receiving a rapid test or a more traditional ELISA. Rapid test, for example, have an accuracy rate exceeding 99%. However, positive results from a rapid or ELISA test must be confirmed by another test to ensure that a person is HIV-positive.

The accuracy of a medical test is a combination of two factors: sensitivity and specificity. The ELISA is extremely sensitive (about 99.5%), which means it will detect very small quantities of HIV antibody. This high sensitivity reduces the odds of reporting a "false negative" when HIV antibodies are present. Assuming you are being tested beyond the "window period" and have not engaged in activities that put you at risk for HIV, if the ELISA is "negative," there is virtually no chance you have HIV.

The high sensitivity of the test creates a slightly lower specificity. This means the result could (infrequently) be "false positive." To compensate for this, confirmatory tests are automatically performed after a positive ELISA. The WB and IFA are highly specific for HIV antibodies, so they rule out false positive ELISAs nearly every time.

The CDC states that the combined accuracy of the ELISA plus either the WB or IFA is greater than 99%.

The CDC recommends retesting any positive (reactive) ELISA twice if either retest is positive (reactive), then a confirmatory test is performed. Only when the confirmatory test is also reactive is the result reported as HIV positive. Again, reputable test sites automatically follow this procedure, so results reported to you as positive can be relied upon completely. It is also important to note that if you test positive through the use of a rapid HIV test (with results provided in 20 minutes or less), your result is still preliminary. A confirmatory test must be performed to verify whether you are infected with HIV and these results will take several days.

What does this mean for you?

  • If you are beyond the window period and were reported HIV negative by a rapid test or an ELISA, and you are not subsequently at risk for HIV, you should consider yourself HIV negative.
    If you want to discuss these issues further -- for example, you want to find out if a certain activity put you at risk for getting HIV -- call an AIDS hotline. Within California, call the San Francisco AIDS Foundation's California AIDS Hotline toll free at 800/367-AIDS. Outside California, call your state's AIDS Hotline or the CDC-INFO toll free at 800/CDC-INFO. To find the number for your state's aids hotline, go to www.aidshotline.org and click on Other AIDS Hotlines.
  • Some individuals, perhaps wanting results without waiting for the window period, may be curious about a PCR or other types of viral load (RNA) testing (testing directly for the virus itself). Viral load testing is generally used to allow physicians to track with great accuracy the progression of HIV in the body -- thus helping HIV-infected patients make choices about appropriate treatment strategies. Most people concerned about their HIV status do not need viral load testing. The antibody test is the cheapest, easiest, and overall most reliable way for individuals to learn their HIV status.

That being said, individuals who have had a recent exposure to HIV and experience symptoms consistent with seroconversion (flu-like symptoms), can request a viral load test from their doctor in addition to the HIV antibody test. This may help identify HIV infection during the window period (before HIV antibodies have developed), though it should be noted that an antibody test is ultimately needed to confirm that one is HIV-infected.

Anonymous HIV Testing

Free anonymous testing is available at Anonymous Test Sites in most counties in California. Anonymous testing means that no one has access to your test results since your name is never recorded at the test site.

Anonymous test sites are preferred by some individuals because:

  • The quality of the education and counseling is usually very good.
  • The testing is usually free.
  • The testing is reliable and automatically includes confirmatory tests.
  • It protects you from any and all risks of discrimination or adverse impact.

People with HIV infection and AIDS are generally reported by name to local and state public health officials (see HIV name reporting section below). Anonymous test results, however, are not reported to the government and can not be because an individual's name is never provided. In addition, anonymous testing sites never give written results. Some sites that provide anonymous testing also provide confidential testing, which may include written results. If you live in California, visit www.aidshotline.org to find sites that offer anonymous or confidential testing or you can call the California AIDS Hotline, toll free, at 1-800-367-AIDS.

At some anonymous test sites, you must make an appointment in others, appointments are not necessary -- you can just drop in. You choose, or are given, a letter/number code that will match you to your test results. Most anonymous test sites provide pre-test counseling and risk assessment. A blood sample is sometimes drawn, but some sites now use rapid HIV testing, which can be done needle-free or utilizes blood taken from a finger-stick. You must return, in person, in about one to two weeks to obtain results, unless the rapid HIV test is used, which can determine your results in less than 20 minutes.

Confidential HIV Testing

HIV antibody testing can be ordered through a private physician, some clinics, and hospitals. If you give your name, even if the test site says your name is known only to them and not the lab, the test is technically confidential, not anonymous. Confidential antibody testing means that you and the health care provider know your results, which may be recorded in your medical file. There are also testing sites that offer confidential testing. Those who are tested confidentially and are found to be infected with HIV are reported to local public health officials so that the government can better track the extent of the disease in the population as a whole.

HIV Name-Based Reporting

California requires those who are infected with HIV to be reported by name to local public health officials. It is important to note that AIDS diagnoses have been reported by name in California and throughout the United States since the early 1980s. The federal Centers for Disease Control and Prevention has urged all states to collect HIV infection reports by name and the vast majority of states now do so.

Fortunately, public health reporting by name has been in place for many years for other communicable diseases and this information has been successfully protected by public health officials. More than 80 other diseases and conditions are currently required to be reported by name in California, including Hepatitis, tuberculosis, syphilis and Chlamydia.

This information is collected to allow public health officials to track these diseases. HIV reporting will make it easier for localities to collect information about the epidemic, providing the government with valid, uniform data for service and prevention planning.

It is important to note that only public health officials have access to the information that is reported employers and insurance companies do not. Additionally, the names of HIV-positive Californians will only be reported to the county in which they live and to the state. The federal government will only receive a unique code for each HIV-positive individual, not their actual name. California law includes provisions that ensure the highest degree of confidentiality of name-based HIV data and significant financial penalties for inappropriate disclosure of that information. In addition, stringent laws exist at the federal, state and local levels to protect individuals with HIV and AIDS from discrimination in housing, employment and public services.

Because public health reporting data are so carefully protected, California's HIV reporting policy should not discourage you from seeking HIV testing and treatment. For those who do have strong concerns about having their HIV status reported to government officials, it may be advisable to seek out HIV testing at an anonymous test site. Because your name is never provided at an anonymous test site, you cannot be reported to the government. It is important to note, however, that if you test positive at an anonymous test site and then seek care for HIV disease from a physician, you will likely be reported by name at that time.

Partner Notification

Voluntary partner notification programs -- also known as partner counseling and referral services (PCRS) -- allow individuals who have tested positive to get help in notifying previous sex or needle sharing partners that they may have been exposed to HIV. Partner notification programs are not new they have been used for years with tuberculosis, syphilis, and other communicable diseases.

Through these services you will receive counseling about how to notify partners yourself or you can have a public health officer do it for you. The health official will ask for the names, addresses, e-mails and/or phone numbers of other people (contacts) with whom you have had sex or shared needles. The health department will then try to locate each contact to tell them that they have been exposed to someone with HIV and could be at risk. They will advise these contacts to be tested for HIV. The health department does not disclose the name of the person who has tested positive, but in some cases, it may be obvious to the contact, particularly if he/she has had sex with only one or few other people.

Orasure Test for HIV

The OraSure HIV antibody test method, approved by the FDA in 1996, uses a sample of oral mucus obtained with a specially treated cotton pad that is placed between the cheek and lower gum for two minutes. Testing oral mucus has also been called "oral mucosal transudate testing." It is similar to the rapid oral test. In fact, the same company makes both products. The only difference between the two is the amount of time that it takes to receive your results.

Note: the saliva and oral mucus contain antibodies to HIV, not HIV itself, so HIV is not transmitted through these fluids.

Some public test sites offer this oral test as an alternative to blood testing.

The sample is sent to a lab, where it undergoes an ELISA procedure. All positive ELISA samples undergo a Western Blot confirmatory test (using the same pad).

The combined accuracy of OraSure ELISA and WB procedures is comparable to traditional blood testing, so it is very high.

Home HIV Antibody Tests

At this time, one company, Home Access, offers an FDA-approved at-home antibody test. This test costs about $45 to $70, depending on whether you pay for 72-hour results (they give you a pre-paid express delivery envelope to expedite shipping your sample to the lab) or standard 7-10 day results.

Home Access uses a blood sample from a finger prick, which you then send to a certified laboratory for testing. You must call a toll-free number to register your sample prior to shipping. Enclosed in the test kit is an identifying number. Results take three days to one week. To obtain results, you give the operator the ID number and they will look up the result of the test. This number system provides you with an anonymous test result so your result cannot be reported to anyone else.

Home Access uses traditional ELISA and Western Blot procedures, so the results are as accurate as one would receive with an antibody test at an anonymous testing site. The privacy of the home test offers some consumers more comfort than going to a public test site.

Remember, as with testing at an anonymous or confidential site, you must wait three to six months (the window period) after your potential exposure to take the at-home test, to be certain if the activity you are concerned about did or did not infect you with HIV.

With the availability of the HIV rapid test, some companies are now considering developing a new type of 'at-home' testing that could provide preliminary test results in less than 30 minutes. Similar to at-home pregnancy tests, consumers would be able get these preliminary results at home without having to mail in a blood specimen (as is required by the Home Access test that is currently available). Such a test has not yet been approved by the FDA. It should be noted that even if it is approved, the person being tested still must wait the 'window period' as previously described, and take a 'confirmatory test' if there is a preliminary positive result.

Urine Test for HIV

A test that detects HIV antibodies in urine was approved by the FDA in 1996. This test is not generally available to consumers. It is usually marketed to insurance companies and to other countries. Note: urine has antibodies for HIV, not HIV itself, so HIV is not transmitted by urine. Manufactured by Seradyn, it is called the "Sentinel" test.

A sample of urine is tested at a certified lab, using a modified ELISA procedure. This test must be ordered by a physician this means the urine HIV test, by definition, is confidential, not anonymous.

Because it is less sensitive than a blood test, positive results must be confirmed by a traditional blood sample (ELISA plus WB).

RNA / NAAT Testing

The "NAT" or "NAAT" test (nucleic acid amplification test) is used to detect the presence of genetic material that is specific to HIV. It is not approved for the use of diagnosis of HIV infection perhaps with the exception of diagnosing infection in newborns of infected mothers who have antibodies to HIV that are transferred from the mother.

However, this test is being increasingly used to detect recent infection at the viral load 'spike' that occurs when HIV is first contracted. This is usually at 2 -- 4 weeks after infection. Although a letter of diagnosis and actual treatment is not given until a three month antibody test is confirmed, this does allow providers to get a patient into health care options sooner. It also lets the client know early on so hopefully they can make risk reducing behavioral choices. It is thought that when this initial infection spike occurs that people are at their most infectious stage, so this test can be used to try and combat that as well.

There is research suggesting that between 10-50% of new infections happen during the acute infection stage. Many feel that identifying and making interventions during this period will greatly reduce infection rates.

A cost-effective and efficient way to combine standard ELISA tests with viral load tests is called a "pooled PCR". Viral load tests are expensive to do individuallys. Instead of performing viral load tests on individual blood samples, they have found a way to combine or "pool" samples and test multiple people's specimens at one time. If a pooled sample tests positive, the laboratory technicians test individual samples in the pool until they find the sample that is positive.

For-Profit Test Sites

There are various for-profit sites that offer HIV testing, but we cannot confirm that they all offer appropriate HIV-related counseling and risk assessment. In addition, many of these companies offer the initial test at a certain publicized fee, but do not tell the consumer that any additional tests (including confirmatory tests) may cost extra.

Most businesses charge a premium because they offer faster results. Remember, standard ELISA tests take only 3.5 hours to do, but most non-profit sites send samples away to central labs to lower costs, which is why their tests are free or low-cost. It is the transit (turnaround) time, not the actual test that takes longer. Some people with high anxiety about exposure may be tempted to use this faster service.

Testing Issues for Specific Populations

Pregnant Women

The number of children reported with AIDS due to perinatal HIV transmission in the United States peaked at 954 in 1992 and declined 95% to 48 in 2004, largely because of the effectiveness of ensuring that pregnant women are encouraged to be tested for HIV and, for those who are infected, to receive treatments that can significantly reduce the risk of transmitting HIV to a newborn. The CDC reports that perinatal transmission rates can be reduced to less than 2% if women are aware that they are infected with HIV and take appropriate treatments to prevent transmission.

Perinatal HIV transmission continues to occur mostly among women who lack prenatal care or who are not offered voluntary HIV counseling and testing during pregnancy. Many of the perinatal HIV infections each year can be attributed to the lack of timely HIV testing and treatment of pregnant women.

However, the antibody test may not be sufficient for a pregnant woman who has had known recent exposure to HIV. In such cases, a viral load test that tests for the actual virus (rather than antibodies) may be ordered by a physician to help the woman make more informed decisions.

Newborns and Children

During pregnancy a mother's antibodies to HIV are transferred to the baby. Therefore, for the first 12-18 months, a child born to an HIV-positive woman will test positive with an antibody test. This does not mean that the child is infected. After 12 to 18 months, the child will shed the mother's antibodies. If he/she is infected, the child will continue to test positive with an antibody test after this period. For this reason, the antibody test is not a reliable indicator of HIV status for children under 18 months. In cases such as these, the viral load test may be used to provide additional information about the child's immune system.

HIV testing may present special problems and situations for children and youth.

  • Children under 12. California's Anonymous Testing Sites will not perform an antibody test on children under 12. Children under 12 have to be tested through a private physician or clinic.
  • Youth 12 to 25 may schedule individual youth appointments at the anonymous testing sites. This means they will receive more personalized counseling from the same counselor before and after the test. See the Referral Database for complete listings of HIV-related services for youth in California (keyword: "adolescents").

Immigrants

[adapted from HIV and Immigrants: A Manual for AIDS Service Providers, published by the National Lawyers Guild with support from the San Francisco AIDS Foundation.]

All applicants for U.S. residence must take an HIV antibody test as part of the compulsory medical exam. Those who test positive are denied residence automatically. This measure also applies to all people requesting change in residence status, including citizenship applications.

Let's say you are an immigrant who has tested HIV positive. Besides worrying about testing HIV positive, you also fear what will happen to you because you are not a U.S. citizen. You may wonder whether your immigration status will change the kind of services you can get. You may ask: Will I be deported? Will I lose my immigration status? Can I work or get public benefits to help me if I need them? Will getting benefits make it hard to get another immigration status? If I can't work or get benefits, is there anything I can do to get another immigration status that will help me?

  • Contact a local immigration assistance agency. For California agencies, go to the Referral Database. Use the keyword "refugee" to find local agencies.
  • Do not go to the United States Citizen and Immigration Services (USCIS), formerly known as Immigration and Naturalization Services (INS), office by yourself. No one should speak to USCIS or go to USCIS before talking to an immigration law expert. If non-citizens go to USCIS by themselves, USCIS may arrest them and remove them from the US before they have had the chance to talk to a lawyer.
  • Become informed about confidential versus anonymous testing. Before taking an INS medical exam, a non-citizen should get tested at a local clinic. Anonymous testing will give them the results without connecting those results to a name and may be safer in these circumstances.

Military Personnel

The Defense Department requires all military personnel on active duty and all members of the reserve and National Guard to be tested for the presence of HIV antibodies at least biennially. Soldiers who receive overseas assignment instructions, or are scheduled for overseas duty that will exceed 179 days, must have tested negative within six months of the departure date.

Soldiers who are infected with HIV will not be deployed outside of the continental United States. Soldiers serving outside the continental U.S who are confirmed as HIV-positive will be expeditiously reassigned to the continental U.S.

The U.S. Army Special Operations Command and Ranger organizations are totally closed to those who are infected with HIV. HIV-positive soldiers may face additional restrictions as determined by the U.S. Department of Defense.

A military doctor can notify the spouse of a reservist, if that reservist has tested positive for HIV.


Importance of HIV Testing for Prevention of HIV Infection

People with HIV who are aware of their status can get HIV treatment (called antiretroviral therapy or ART) and remain healthy for many years. Studies show that the sooner people start treatment after diagnosis, the more they benefit from ART. Treatment with ART reduces the amount of HIV in the blood (called viral load), reduces HIV-related illness, and helps prevent transmission to others. People with HIV who take HIV medicine as prescribed and get and keep an undetectable viral load (or stay virally suppressed) have effectively no risk of transmitting HIV to HIV-negative sex partners.

People who get tested and learn they don&rsquot have HIV can also make decisions about sex, drug use, and health care that can protect them from HIV. For people at risk for HIV, taking HIV medicine called pre-exposure prophylaxis (or PrEP) is highly effective for preventing HIV.


Major Viral Infections

In this review, we focus on HIV, hepatitis C virus (HCV) and HPV because these three major viruses are responsible for a series of worldwide epidemics that have had an enormous effect on morbidity and mortality. Most people now understand the impact and risk of HIV infection, but the risk and sequelae of HPV and HCV infections are much less recognized. Recently, the Centers for Disease Control and Prevention (CDC), Atlanta, reported that HCV is responsible for more deaths in the United States than is HIV. 38 Furthermore, HPV, originally associated only with cervical cancer, now is linked to an increasing incidence of oral cancer.

Salivary diagnostics for HIV

HIV is the cause of AIDS. The infection of immune system cells eventually leads to the loss of cell-mediated immunity. If the infection is left untreated, opportunistic infections and cancers develop, which eventually lead to death. HIV infection is detected easily with an antibody-based screening test after seroconversion however, early infections are difficult to recognize because they are accompanied only by mild flulike symptoms, and an antigen or nucleic acid assay is required in the weeks before seroconversion. Diagnosis according to a reactive antibody assay requires a confirmatory test with either a Western blot (via blood or saliva) or a polymerase chain reaction (PCR) (via blood). Well-managed drug therapy is required to keep viral propagation at close to undetectable levels. Currently, no cure exists for HIV infection, and once it is integrated into the human genome, it remains and can replicate unless suppressed by medication. Although some investigators have reported the isolation of infective viral particles from oral samples and demonstrated the presence of viral particles in epithelial cells of the buccal mucosa, 39-41 the chance of transmitting HIV through saliva remains extremely low. 42 Moreover, a large body of literature supports the presence of effective anti-infective activity of human salivary secretions by a variety of salivary proteins, including defensins, lysozymes, lactoferrin secretory leukocyte protease inhibitor and DMBT1 (glycoprotein-340/salivary agglutinin), 43 as well as lysis of HIV in the oral cavity owing to the hypotonicity of saliva. 44

All of the existing oral-based diagnostic tests for HIV infection are screening tests, detecting antibodies to HIV-1 or both HIV-1 and HIV-2. In general, these tests involve the use of nitrocellulose lateral flow strips that contain two capture zones: a control line that detects the presence of all antibodies in the sample and a test line that specifically reacts with HIV-1 or, ideally, with both HIV-1 and HIV-2. A reactive result needs to be confirmed with a second test. This confirmatory test can be a Western blot that involves the use of saliva or blood and that detects antibodies to multiple HIV antigens, or it could be a blood-based PCR test that detects HIV RNA.

Although many oral tests are on the market, the U.S. Food and Drug Administration (FDA) has approved only one test. The test, which was approved in 2004, 45 involves use of a POC device (OraQuick ADVANCE Rapid HIV-1/2 Antibody Test, OraSure Technologies, Bethlehem, Pa.). The clinician collects oral fluid with a swab and places it directly into a developing solution in the device after 20 minutes, he or she can visualize the resulting lines.

Results from multiple studies demonstrated that the sensitivity and specificity of these oral tests are comparable to those of tests for antibody detection that involve the use of plasma or finger-stick blood. 46.47

Several investigators have conducted studies pertaining to the development and application of technologies used to detect HIV antibodies, HIV-derived antigen and nucleic acids in oral samples. 3,15,48-58 These include technologies used for high-throughput tests conducted in clinical laboratories, as well as rapid, single-sample tests for POC or home-testing devices. As is seen for other infectious diseases, salivary antibody diagnostics for HIV are as effective as blood-based diagnostics. However, because of differences in concentration and stability, other pathogen-specific targets (antigen, nucleic acid) are not always detectable in saliva.

For example, the fourth-generation immuno-assays detect p24 antigen and antibodies against HIV, allowing earlier detection of HIV infection with blood-based samples. 59,60 However, investigators have not yet demonstrated that these tests work with saliva samples. Similarly, detection of viral RNA in saliva is more difficult than is detection in a blood sample owing to decreased viral load. Researchers have reported higher loads of HIV in saliva than in serum in some patients, 61 and these patients are referred to as hypersecretors. Detection of HIV RNA in saliva is possible because current technologies include concentration and purification steps to attain the required sensitivity.

Salivary diagnostics for HCV

The common hepatitis viruses are named with the letters A through E. Vaccines are available for hepatitis A virus and hepatitis B virus (HBV) vaccines are in development for hepatitis E virus, but the FDA has not yet approved them. Blood safety procedures for donor blood for transfusion-transmissible infectious diseases include various tests for HBV (screening for the presence of antibody and antigen) and HCV (screening for the presence of antibody and nucleic acid targets). No vaccine currently is available for HCV.

HCV, like HIV, is an RNA virus. Chronic infection causes liver cirrhosis, which may lead to liver failure, cancer or extremely dilated sub-mucosal veins in the stomach and esophagus. Acute infections generally are accompanied by mild symptoms and are not recognized easily. In contrast to HIV, HCV infections can resolve spontaneously however, like HIV, the virus may remain latent and can be activated at a later time. The first step in screening is to test for the presence of antibodies if the test result is positive, then a confirmatory test is required. Typically, the confirmatory test, as for HIV, is a Western (immunoblot) assay combined with a nucleic acid�sed viral load assay. 62

Recently, there has been a great deal of interest in saliva-based rapid tests for HCV, which has been referred to as the “silent epidemic.” 63 As mentioned earlier, the CDC recently reported that more people in the United States die each year of HCV than they do of HIV. 38 Because a number of drugs are available to treat HCV, and many more are under development, diagnostic testing for the presence of the virus can lead to timely therapeutic intervention. Screening tests for HCV, similar to those for HIV, typically rely on detecting specific anti-HCV antibodies. Although an Internet search reveals several such tests, none of these saliva-based tests, to date, has received FDA approval.

OraSure Technologies markets an FDA-approved test for HCV that uses finger-stick blood in addition, the company has a salivary test that is used widely in Europe but has not been approved for sale in the United States. Drobnik and colleagues 64 compared screening test results of the OraQuick HCV Rapid Antibody Test (OraSure Technologies) with those of the criterion standard enzyme immunoassay (by means of a blood draw) (Abbott HCV EIA 2.0, Abbott Laboratories, Abbott Park, Ill.). The researchers confirmed reactive samples with use of a Western blot (Chiron RIBA HCV, Bio-Rad Laboratories, Hercules, Calif.). The authors reported that the test results of the OraQuick HCV Rapid Antibody Test matched those of the Abbott HCV EIA 2.0 immunoassay 97.5 percent of the time. 64

Salivary diagnostics for HPV

HPV is a DNA virus investigators have identified more than 100 HPV types, 65 approximately 20 of which are considered to be high-risk HPVs with the possibility of leading to malignant neoplasia. About 70 percent of cervical cancers are attributed to HPV types 16 and 18 and are part of a national prevention and screening program that includes use of an anti-HPV vaccine. Most HPV infections are self-cleared but antibodies to the virus remain thus, antibody tests to screen for HPV infection are not considered useful.

Since 1990, there has been a striking increase in oropharyngeal squamous cell carcinoma (OSCC), and approximately 60 percent of these tumors are associated with HPV. 66 These tumors largely are seen in white men with no history of tobacco or alcohol use, in contrast to oral cancer not associated with HPV, in which tobacco and alcohol use are major factors in disease development. Chaturvedi and colleagues 67 reported that although the incidence of HPV-negative OSCC decreased from two cases per 100,000 to one case per 100,000 from 1988 to 2004, HPV-associated OSCC increased from 0.8 case per 100,000 to 2.6 cases per 100,000 during the same period.

Gillison and colleagues 68 conducted a large study of the prevalence of oral HPV infection in the United States in 2009-2010. They reported that oral HPV infection in men and women aged 14 through 69 years was 6.9 percent, with a 1 percent incidence of high-risk HPV 16. An intriguing HPV genome sequencing report by Andrews and colleagues 69 pertaining to two couples who developed HPV-associated tonsillar carcinoma revealed the identical HPV 16 in the partners of both couples, which suggests the potentially infectious nature of this cancer.

Salivary diagnostic tests are available for HPV, and essentially they involve the use of PCR thus, they are not POC tests. Kits containing a salivary collector are placed in transport media and sent to a central laboratory for analysis. Investigators in the field have used oral swabs, expectorated saliva or an expectorated oral rinse with mouthwash (OraRisk HPV test, OralDNA Labs, Brentwood, Tenn., which, to our knowledge, is the only salivary diagnostic test for HPV commercially available in the United States). 70,71 The latter collection technique probably has the highest sensitivity, because it samples the entire oral cavity and the swishing of the solution dislodges mucosal cells. Investigators in the laboratory use a variety of primers to detect as many HPV types as possible. Early diagnosis is critical for survival of patients with OSCC, and, thus, it is likely that use of salivary HPV analyses will continue to increase.


Newly identified antibody can be targeted by HIV vaccines

A newly identified group of antibodies that binds to a coating of sugars on the outer shell of HIV is effective in neutralizing the virus and points to a novel vaccine approach that could also potentially be used against SARS-CoV-2 and fungal pathogens, researchers at the Duke Human Vaccine Institute report.

In a study appearing online May 20 in the journal Cell, the researchers describe an immune cell found in both monkeys and humans that produces a unique type of anti-glycan antibody. This newly described antibody has the ability to attach to the outer layer of HIV at a patch of glycans -- the chain-like structures of sugars that are on the surfaces of cells, including the outer shells of viruses.

"This represents a new form of host defense," said senior author Barton Haynes, M.D., director of the Duke Human Vaccine Institute (DHVI). "These new antibodies have a special shape and could be effective against a variety of pathogens. It's very exciting."

Haynes and colleagues -- including lead author Wilton Williams, Ph.D., director of the Viral Genetics Analysis Core at DHVI and co-author Priyamvada Acharya, Ph.D., director of the Division of Structural Biology at DHVI -- found the antibody during a series of studies exploring whether there might be an immune response targeted to glycans that cover the outer surface of HIV.

More than 50% of the virus's outer layer is composed of glycans. Haynes said it has long been a tempting approach to unleash anti-glycan antibodies to break down these sugar structures, triggering immune B-cell lymphocytes to produce antibodies to neutralize HIV.

"Of course, it's not that simple," Haynes said.

Instead, HIV is cloaked in sugars that look like the host's glycans, creating a shield that makes the virus appear to be part of the host rather than a deadly pathogen.

But the newly identified group of anti-glycan antibodies -- referred by the Duke team as Fab-dimerized glycan-reactive (FDG) antibodies -- had gone undiscovered as a potential option.

To date, there was only one report of a similar anti-glycan HIV antibody with an unusual structure that was found 24 years ago (termed 2G12). The Duke team has now isolated several FDG antibodies and found that they display a rare, never-before-seen structure that resembled 2G12. This structure enables the antibody to lock tightly onto a specific, dense patch of sugars on HIV, but not on other cellular surfaces swathed in host glycans.

"The structural and functional characteristics of these antibodies can be used to design vaccines that target this glycan patch on HIV, eliciting a B-cell response that neutralizes the virus," Williams said.

"These antibodies are actually much more common in blood cells than other neutralizing antibodies that target specific regions of the HIV outer layer," Williams said. "That's an exciting finding, because it overcomes one of the biggest complexities associated with other types of broadly neutralizing antibodies."

Williams said the FDG antibodies also bind to a pathogenic yeast called Candida albicans, and to viruses, including SARS-CoV-2, which causes COVID-19. Additional studies will explore ways of harnessing the antibody and deploying it against these pathogens.

In addition to Haynes, Williams and Acharya, study authors include R. Ryan Meyerhoff, R.J. Edwards, Hui Li, Kartik Manne, Nathan I. Nicely, Rory Henderson, Ye Zhou, Katarzyna Janowska, Katayoun Mansouri, Sophie Gobeil, Tyler Evangelous, Bhavna Hora, Madison Berry, A. Yousef Abuahmad, Jordan Sprenz, Margaret Deyton, Victoria Stalls, Megan Kopp, Allen L. Hsu, Mario J. Borgnia, Guillaume Stewart-Jones, Matthew S. Lee, Naomi Bronkema, M. Anthony Moody, Kevin Wiehe, Todd Bradley, S. Munir Alam, Robert J. Parks, Andrew Foulger, Thomas Oguin, Gregory D. Sempowski, Mattia Bonsignori, Celia C. LaBranche, David C. Montefiori, Michael Seaman, Sampa Santra, John Perfect, Joseph R. Francica, Geoffrey M. Lynn, Baptiste Aussedat, William E. Walkowicz, Richard Laga, Garnett Kelsoe, Kevin O. Saunders, Daniela Fera, Peter D. Kwong, Robert A. Seder, Alberto Bartesaghi and George M. Shaw.

The study received funding support from the Consortia for HIV/AIDS Vaccine Development, the National Institutes of Health, the National Institute of Allergy and Infectious Diseases, Division of AIDS (UM1-AI44371).


What are the Western blot and ELISA tests for HIV?

The Western blot and ELISA tests are two blood antibody tests that may be used to detect HIV.

In the past, the Western blot test was used to confirm the results of an ELISA test.

However, advances in technology mean that other methods are now commonly used. Since 2014, the Centers for Disease Control and Prevention (CDC) have recommended discontinuing the Western blot test.

Now, most laboratories use an immunoassay for the HIVp24 antigen and antibodies to HIV-1 and 2, followed by a confirmatory immunoassay to distinguish between HIV-1 and HIV-2.

Testing and diagnosis are an important part of staying healthy with HIV. With early diagnosis, early treatment is possible. Testing is the first step in accessing effective ways of managing the condition. It is the key to both treatment and prevention.

Current treatment can reduce the viral load to undetectable levels. While levels are this low, the body can remain healthy, the individual can expect a normal lifespan , and the virus cannot be transmitted.

Share on Pinterest The ELISA test is a blood antibody test that checks for proteins the body makes if HIV is present.

Laboratory blood tests can be used to diagnose HIV through detecting certain antibodies or proteins produced by the immune system in response to the virus.

The ELISA test, also called the EIA for enzyme immunoassay, is used to detect the HIV antibody. It checks for certain proteins that the body makes in response to HIV.

The blood sample will be added to a cassette that contains the viral protein, called antigen.

If the blood contains antibodies to HIV, it will bind with the antigen and cause the cassette’s contents to change color. This very sensitive test was the first one widely used to check for HIV.

The Western blot test was previously used to confirm the result of the ELISA, but it is no longer recommended, as other tests are now more reliable and enable a faster diagnosis.

In the Western blot test, the blood is taken in the same way, but the sample is separated with an electrical current and transferred onto a piece of blotting paper. Here, an enzyme is added to cause color changes that signal the presence of HIV antibodies.

Who has the tests?

Most adults will undergo screening at some time. It is a routine procedure during pregnancy.

However, the Western blot and ELISA tests are only recommended if a person may have been exposed to HIV.

People with a high risk of exposure include:

  • those who have sex without using a condom, especially with someone who has HIV
  • those who share needles
  • people who had blood transfusions or injections prior to 1985
  • those with other sexually transmitted diseases (STDs)

Some people choose to get tested for HIV fairly regularly, for example, if they have a new sexual partner or work in healthcare situations.


Part 2. Full Text of Announcement

More than 38 million individuals worldwide are currently living with HIV new infections occur at a rate of 1.7 million per year worldwide, and 39,000 per year in the U.S. Although currently there is no cure for HIV, current antiretroviral treatment (ART) regimens are very potent and reduce plasma virus to undetectable levels in most patients, leading to reduced morbidity, mortality, and transmission. Multiple clinical studies have provided strong evidence that Undetectable = Untransmittable (U=U) — people who maintain an undetectable viral load cannot sexually transmit the virus to others. However, at least 20% of HIV-positive individuals are unaware of their HIV status, and nearly 40% are not on suppressive treatment. For individuals who do achieve undetectable HIV levels in blood, viral rebound can occur following inconsistent adherence to the treatment regimen, intentional treatment interruption, or emergence of drug resistance mutations. The timing of rebound can be unpredictable, usually with a rapid increase in viral load. Regular self-monitoring for HIV would help individuals identify viral rebound quickly and seek intervention to prevent clinical progression and transmission.

Since 2016, the World Health Organization has recommended HIV self-testing as an additional approach to increase HIV testing services. Currently available rapid HIV self-tests depend on detection of host antibody responses, which are most reliably detectable several weeks after infection. These tests could not be used to detect the initial peak viremia in early infection, before antibody production, nor viral rebound following treatment interruption or emergence of drug resistance, where antibodies would already be present prior to rebound. Direct detection of HIV, rather than antibody, would be necessary in these cases. Sensitive and accurate tests to directly measure HIV involve either expensive, sophisticated equipment in a lab or hospital setting, or point of care devices that require a trained individual to administer the test and read the result. Development of simple, inexpensive, rapid self-testing assays to directly detect HIV would enable individuals to more easily monitor their HIV or viral suppression status. Rapidly evolving molecular diagnostics and manufacturing technologies could be useful in developing innovative approaches to fulfilling this need.

The purpose of this Funding Opportunity Announcement (FOA) is to stimulate innovative, collaborative, interdisciplinary research and early-stage diagnostic technology development focused on rapid, simple, sensitive, and cost-effective assays appropriate for HIV self-testing during the earliest phases of acute infection and/or during viral rebound. The proposed research should establish feasibility for direct detection of HIV (RNA, DNA, protein, or biomarkers that reliably correlate with HIV positivity or rebound) in a qualitative or semi-quantitative diagnostic self-test assay. The assays should be designed with a user-centered approach and should be as easy to perform as a home pregnancy test or an in-home glucose monitoring device.

Research Objectives and Scope

This FOA will support high-risk, bi-phasic, milestone-driven research and early-stage development of innovative technologies designed to enable rapid self-testing assays to meet one or both of the following research objectives:

  • detecting HIV (RNA, DNA, protein or biomarker) at the earliest stage of initial infection, ideally less than 2 weeks post-infection and/or
  • detecting HIV rebound in treated individuals as early as possible following treatment interruption or loss of viral suppression by ART.

Strategies may include either the initial development of a novel technology or adaptation of an existing technology to detect HIV from finger stick blood or other biospecimen. Proposed studies are expected to be at the discovery and feasibility stage, designed to show potential efficacy in the detection of HIV and feasibility as a self-test platform.

Proposed studies should emphasize innovation and exploratory research with a focus on qualitative or semi-quantitative HIV detection and assay development. Innovative technologies such as microfluidics for electricity-free RNA/DNA amplification, synthetic biology approaches, paper- or plastic-based analytical platforms, and smartphone-enabled diagnostics are highly encouraged. Additional studies to address assay feasibility may include but are not limited to:

  • comparison of multiple related detection strategies
  • development and integration of sample preparation technologies
  • development and integration of readout technologies
  • evaluation of different biospecimens for virus detection
  • evaluation of biomarkers for reliable correlation with HIV infection and/or rebound

Development of such technology is expected to require collaboration among experts in the fields of virology and biotechnology (e.g., microfluidics, bioengineering, synthetic biology, nanotechnology, and/or manufacturing), as well as clinical, social and behavioral science. Applicants must demonstrate that they are capable of developing the proposed technologies and assay, have access to appropriate virus and biospecimen resources, and have the means to obtain meaningful end-user input on assay performance and usability during early-stage assay development.

Upon completion, successful projects will have demonstrated proof-of-concept for prototype assay technology capable of detecting HIV in human biospecimen samples without the use of expensive equipment or trained individuals. Assays ready for more advanced product development and commercialization would be potentially appropriate for the NIAID SBIR/STTR program (https://www.niaid.nih.gov/research/grants-small-business).

Specific performance criteria are not required, but assay design, proposed studies, and milestones for feasibility should consider how to achieve desirable features for an ideal diagnostic:

  • Rapid: The test should have a short target diagnostic time to final result.
  • Culture-independent: The test should allow direct detection of HIV or other biomarkers from human samples without relying on cell culture.
  • Accurate and sensitive: The test should accurately identify HIV when the plasma viral load is at levels appropriate for early detection (e.g. equivalent to 1000 to 5000 copies/ml).
  • Specific: The assay should be able to detect any major circulating HIV clades in the target population with high specificity. For monitoring viral suppression, the assay should appropriately consider the need to prevent detection of cell-associated HIV DNA and RNA.
  • Easy to use: for example, an integrated, closed sample-to-answer system with simple, automatic analysis and/or result presentation that does not rely on laboratory facilities or trained individuals to administer or interpret results.
  • Acceptability: for example, themes such as fit with lifestyle or routine activities, no or limited side effects, attitudes toward test, and confidentiality and privacy concerns.
  • Refrigeration-independent: The diagnostic should be designed to operate in a normal range of ambient temperature without need for cold storage.
  • Minimally dependent on electricity: The diagnostic should be designed to operate either without electricity or with a simple battery. Readout devices requiring a cell phone would be acceptable.
  • Cost-effective: Anticipated production and operating costs should be as inexpensive as possible so as to be affordable and practical for repeated testing by the end user.

NIDA is interested in the following research areas relevant to people who inject and/or use drugs (PWID/PWUD) who are likely to experience gaps in HIV testing and care:

  • Studies that address feasibility as it applies to substance using populations living with HIV (i.e., easy to self-administer, cost effective, and potential for mail-in testing and follow up through mobile technologies)
  • Studies focused on developing technologies with sensitive and rapid read out
  • Studies that include plans for post-administering surveys to assess rates of access, acceptability (stigma and ethical concerns), accuracy, and treatment outcomes
  • Studies proposing technologies that are easily adaptable for implementation in homeless shelters, community clinics, criminal justice and emergency department settings

NICHD is interested in the following research areas:

  • Research on the discovery of novel and innovative self-test assays optimized for use in infants, children, adolescents and pregnant as well as non-pregnant women to detect the earliest stage of initial HIV infection (ideally less than 2 weeks post-infection) and/or detect HIV rebound in treated individuals as early as possible following treatment interruption or loss of viral control
  • Formative studies of self-test assays that also explore the feasibility, acceptability and use patterns of self-testing in infants, children, adolescents and pregnant as well as non-pregnant women and the consequences of regular self-test use (e.g. appropriate end users, fit with life routines, testing attitudes and unintended uses like serosorting, confidentiality and privacy issues)

Applications Not Responsive to this FOA

Applications including any of the following will be considered non-responsive and withdrawn prior to review:

  • Focus on detection of SIV or pathogens other than HIV-1
  • Studies focused on detection of host antibody responses or CD4 T cell levels as biomarkers for infection
  • Applications that are focused exclusively on sample preparation, target enrichment, and/or readout without substantial emphasis on virus or biomarker detection
  • Proposal of an assay that is not designed to be a self-testing assay or that requires a trained technician, laboratory setting, or sending of self-collected samples to a separate facility
  • Advanced product development, scale-up manufacturing, and commercialization of assays beyond the discovery, feasibility, and proof-of-concept stages for HIV detection
  • Applications that do not include milestones
  • Applications that do not include aims for both R61 and R33 phases
  • Applications proposing only the R61 phase or only the R33 phase
  • Studies including vertebrate animal models
  • Clinical trials

Due to the high-risk, high-impact nature of the research, this funding opportunity will use the R61/R33 phased innovation grant award mechanism. Support will be provided for up to three years (R61 phase) for hypothesis- and milestone-driven basic technology research, assay development, and end-user input. Up to two years of support may follow (R33 phase) for additional activities as appropriate, such as expanded assay development, optimization, proof of concept validation with human samples, and usability testing. Proposed milestones will be reviewed and negotiated prior to award.

Before the end of the R61 phase, awardees will submit the R33 transition package, which includes a detailed progress report describing advancement toward the initial milestones and a description of how the completed work justifies continuation with the originally proposed R33 studies. These materials will be evaluated by NIH Program staff grants selected for continued funding will be transitioned to an R33 award without the need to submit a new application. Transition to the R33 phase is neither automatic nor guaranteed R33 funding decisions will be based on the original R61/R33 peer review recommendations, successful completion of transition milestones, Program priorities, and availability of funds. It is expected that approximately one-half of the projects supported during the R61 Phase will continue into the R33 Phase.

See Section VIII. Other Information for award authorities and regulations.


HIV Diagnostic and Molecular Testing

Antibodies to HIV-1 and HIV-2 are detected by enzyme immunoassay (EIA). Reactive results are confirmed by HIV-1 Western blot. The HIV Western blot identifies antibodies against eight HIV-1 encoded proteins: p18, p24, p31, gp41, p51, p55, p65/66, gp120/p160. Criteria accepted by CDC/ASTPHLD are used for determining a positive HIV Western blot. These criteria require antibodies against any two of the following HIV-1 proteins: p24, gp41, gp120/160. Specimens showing reactivity to HIV-1 protein(s), but not fulfilling the criteria for a positive result, are reported as Indeterminate. All indeterminate Western blots are further tested in supplemental HIV-1 and HIV-2 specific assays. Specimens showing reactivity to non HIV-1 proteins are not assigned an indeterminate status but are instead reported as “Antibody to non-HIV-1 encoded proteins”. A negative Western blot has no detectable bands, i.e. no antibodies reacting to either HIV-1 or non-HIV-1 proteins. Supplemental assays are performed on EIA-reactive specimens which do not confirm by HIV-1 Western blot. Specimens may be forwarded, upon request, for HIV-2 specific Western blot if supplemental assays indicate HIV-2 antibodies may be present. HIV-1 and -2 EIA screens are run daily, Monday through Friday. HIV-1 Western blot confirmation assays are run on Tuesday, Thursday and Friday.

See Human Immunodeficiency Virus 1 & 2 in the Online Guide to Lab Testing.

HIV-1 RNA Quantitation

Quantitation of HIV RNA copy number is available to monitor antiviral therapy and to predict disease progression in HIV infected persons. HIV RNA quantitation may be useful to indicate when an HIV infected person should start anti-retroviral therapy and when such therapy should be adjusted. (Alone, this assay is not recommended nor approved for diagnosing HIV infection. However, in conjunction with a positive DNA PCR or a reactive EIA, the RNA quantitation may be diagnostic.) High levels of RNA are found during acute infection and in patients who are more likely to have disease progression. Inhibition of cell-free HIV, as reflected by RNA copy number, is associated with better CD4 response and clinical response in some patient populations.

To quantify HIV RNA, the UW Clinical Retrovirology Laboratory uses a real-time reverse transcription (RT)-polymerase chain reaction (PCR) amplification platform with enhanced sensitivity and broader dynamic range compared to available commercial assays. The Real-time RT-PCR assay for HIV RNA quantification has been validated against the commercial bDNA and ultrasensitive (US) RT-PCR assays.

*The dynamic range for HIV RNA detection by Real-Time RT-PCR is 30 to 1,000,000 copies/mL of plasma.

HIV clade B is the predominant virus causing HIV/AIDS in North America and Europe. To provide HIV RNA quantification for non-clade B viruses, the Roche Monitorª US-RT-PCR version 1.5 assay should be ordered.

*The dynamic range for HIV RNA quantitation by US-RT-PCR is 50 to 100,000 copies/mL of plasma.

See HIV-1 RNA Quant by Real Time in the Online Guide to Lab Testing.
See HIV-1 Quantitative RNA by US rt PCR in the Online Guide to Lab Testing.

HIV-2 RNA Quantitation

The Retrovirology Laboratory within the University of Washington Department of Laboratory Medicine offers a new algorithm for the laboratory diagnosis of HIV using a fourth generation HIV screening assay (Abbott Architect HIV Ag/Ab Combo Assay) and an HIV-1/-2 differentiation assay (Bio-Rad Multispot HIV-1/HIV-2 Rapid Test) to confirm the presence of HIV-1 or/and HIV-2 antibodies. Acute HIV-1 infections are confirmed with the HIV-1 RNA assay (Abbott RealTime HIV-1 assay). HIV-1 RNA and HIV-2 RNA assays are offered to confirm HIV-1 or/and HIV-2 infections for patient samples with undifferentiated HIV antibody status and to monitor antiretroviral therapy.

The HIV-2 RNA Quantitative Viral Load assay (HIV2VL) was developed and validated according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. Recommended samples include plasma (EDTA) and CSF. The HIV2VL has successfully tested patients’ samples nationwide and from Canada since 2011.

HIV-1 Proviral DNA Detection

The detection of cell associated Human Immunodeficiency Proviral DNA by polymerase chain reaction (PCR) amplification is one of the most sensitive non-serologic methods for confirming HIV infection. In addition to HIV culture, this assay is recommended for confirming HIV infection in the neonate. HIV DNA PCR may also be used as a supplemental test to determine the significance of an indeterminate HIV Western Blot serology result.

See HIV-1 Proviral DNA by PCR in the Online Guide to Lab Testing.

HIV-1 Culture

Culture is an extremely sensitive virologic method for documenting HIV infection, especially in neonates whose serologies are complicated by the presence of maternal antibody. Since cultures must be processed within 30 hours of collection, specimens should not be obtained Friday PM through Sunday AM.

See HIV-1 Qualitative Culture in the Online Guide to Lab Testing.
See HIV-1 Quantitative Culture in the Online Guide to Lab Testing.

HIV-1 Genotypic Resistance Assay

This assay is performed once a week and an interpretive report is sent (see example of PDF document linked below). The assay involves sequencing of the HIV pol gene, after which mutations in the gene can be compared to sequences known to confer resistance to different classes of antiretroviral drugs. The assay is most useful in patients who lose viral suppression on antiretroviral therapy and should be performed before switches in therapy are entertained. Specimen requirement is a 10 mL EDTA tube at room temperature or frozen plasma at -70°C, shipped on dry ice, is also acceptable.