Despite extensive testing possibilities and recommendations HIV infection continues to be recognized and diagnosed more often than not at a later stage. At the time of initial diagnosis approximately one third of HIV patients already have an immunodeficiency with a CD4 T cell count below 200/μl. In half of these late-diagnosed infections an AIDS-related illness is simultaneously diagnosed (RKI 2007). Rapid diagnosis of HIV infection is also important in order to avoid further transmission. Every pregnant woman should be offered an HIV test. HIV testing also plays an important security role in blood and organ donation.
The basics of HIV diagnostics
The diagnosis of HIV infection is primarily based on a laboratory screening test. A reactive result of a screening test has to be confirmed by an alternative assay (confirmatory test). Due to its relatively high sensitivity, the 4th generation test (“Combo test”) which simultaneously detects both HIV-specific antibodies and p24 antigen should be used (Breast 2000, Weber 2002, Sickinger, 2004, Skidmore 2009). A “seronegative” chronic HIV infection is an absolute rarity and irrelevant in practice (Spivak 2010). Any approved screening test detects all known HIV types (HIV-1 and -2), HIV groups and HIV subtypes.
There are numerous commercial systems available for screening. However, the basic technological principle is the same for all and is based on antigen-antibody binding. The prototype assay is the ELISA (enzyme linked immunosorbent assay). Its central element is a plastic plate with 96 wells (microtiter plate). The surface of each cavity is coupled with HIV antigens and HIV antibodies. When a patient’s serum or plasma containing HIV antibodies is placed into one cavity, antibodies bind to the coupled antigen. An enzyme-labelled second antibody is then added, which recognizes and binds to human antibodies. Finally a substrate is added that is converted by the enzyme at the second antibody. The result is a colour change, measured photometrically. The optical density correlates with the HIV antibody concentration in the sample of the patient – the higher the intensity, the more antibodies present in the sample.
Based on this prototype several advances have improved the efficiency and effectiveness of the screening test (Perry 2008). Modern test systems are highly automated to achieve a very high degree of standardization and generate a result in significantly less than one hour. In these systems, the solid phase consists of microparticles coupled with the virus antigens and antibodies. Accordingly, the method is referred to as a “microparticle enzyme immunoassay” (MEIA).
The measured value is usually an index without dimensions, calculated from the ratio of the measured value of the patient sample and the negative control (Sample/Control, S/Co). Values below 1 are considered negative, values above 1 as reactive. It should always be called “reactive” and not a “positive” result to document that this result needs to be confirmed by a second test.
With the screening test, sensitivity has the highest priority (this way, no infection should be missed), while a high specificity is preferred for the confirmatory test. Screening tests approved in Germany require a specificity of 99.5%. That means that one in 200 HIV-negative samples could have a false-reactive test result. False-reactive results are caused for example by stimulation of the immune system (e.g., viral infections, pregnancy, vaccinations, autoimmune diseases). Thus, in certain patient groups (e.g., pregnant women, dialysis patients) an increased proportion of false reactive test results can occur.
To confirm a reactive screening test result a Western Blot (immunoblot) analysis is typically carried out. Viral proteins (antigens) are separated by their molecular weight via electrophoresis and transferred to a membrane, which is then used as a test strip. An advancement in terms of standardization is the so-called line blot which is produced by spraying recombinant HIV antigens directly onto a test membrane. The test strip is incubated with the serum or plasma of the patient. If HIV-specific antibodies are present, they bind to the antigen. Analogous to the ELISA (see above) the resulting antigen-antibody complex will become visible on the test strip using an enzyme-labeled second antibody and a corresponding substrate. According to the antibody specificities present in the sample a corresponding band spectrum occurs on the test strip.
The various HIV proteins are assigned to three functional groups (“p” – protein, “gp” – glycoprotein. The numbers refer to the molecular weight):
● envelope proteins (env): gp41, gp160, gp120
● polymerase proteins (pol): p31/p34, p39/p40, p51/p52, p66/p68
● core proteins (gag): p17/p18, p24/p25, p55
The formation of antibodies after infection follows a specific kinetic: while p24 and gp120 antibodies are detectable early, the p31 band usually occurs later in the course of infection (Fiebig 2003). A Western Blot is considered positive when at least two or three bands are visible. With regard to the antibody specifics, the criteria for a positive result are internationally not uniformly defined. According to the German guidelines, based on the DIN 58 969 Part 41 (“serodiagnosis of infectious diseases – immunoblot”), a test result is considered positive when antibodies against an env protein and also against a gag protein and/or a pol protein are detected. According to WHO criteria a Western Blot is positive when antibodies against at least 2 env proteins are detectable. According to the guidelines of the American Red Cross a gag, pol and env-band is required. The US FDA demands a p24, a p34 and a gp41 or gp120/160-band. For example, a Western Blot with a gp120 and p24 band would be interpreted borderline according to the WHO and positive to the German criteria. A weak band spectrum may indicate an early phase of an HIV infection and further tests such as PCR should be carried out (see below).
Compared to a 4th generation screening test the p24 antigen is not included in the confirmatory test. In the case of “reactive screening test – negative confirmatory test”, acute HIV infection where HIV-specific antibodies are not yet formed although the p24 antigen is present cannot be excluded. Such a result should be checked after 2-3 weeks. If a patient is concerned regarding an acute infection (acute retroviral syndrome, recent exchange of bodily fluids with an HIV-infected person) the implementation of an HIV PCR is useful. The PCR is also recommended in case of a highly positive screening and negative confirmatory test result. It is recommended to consult the laboratory to discuss the adequate procedure.
Ideally, the laboratory will use a Western Blot, which also covers antibodies for HIV-2. In general, a synthetic HIV-2 peptide is used for this purpose. In case of a reactive HIV-2 band, this result must be confirmed by an HIV-2-specific Western Blot.
As an alternative to the Western Blot for confirmation of a reactive screening test an immunofluorescence assay (IFA) is available, although less common.
To exclude sample confusion each first positive test result should be confirmed by examination of a second sample. If a patient is suspected to have an HIV infection, the result of viral load measurement can be used for confirmation (see chapter on HIV monitoring). In this case, a second serological test is not necessary.
In addition to the serological test systems, molecular methods for detection of HIV RNA (PCR, bDNA) are available. The quantitative detection of HIV RNA (a viral load determination) is one of the essential components of the monitoring of HIV infection (Berger 2002, Wittek 2007).
In the context of primary HIV diagnosis however, HIV PCR, is reserved only for specific issues such as the suspicion of acute infection or vertical transmission (see below). For the general exclusion of HIV transmission, HIV PCR is only conditionally suitable and cannot replace the serological HIV test. Furthermore, the commercially available test systems have not been validated yet by the manufacturers for primary diagnosis.
Rapid HIV tests functionally correspond to a screening test, i.e., a reactive result must be confirmed by a Western Blot analysis. Rapid tests can be carried out quickly, easily and without any equipment expense and can therefore be used as so-called “point of care” tests. In addition to plasma and serum, full or capillary blood (from the fingertip or the ear lobe) is suitable as test material, so that no centrifuge is required. In some test systems urine or oral transsudate (not saliva) may be used. However, rapid tests exhibit less sensitivity if specimens others than serum or plasma are used (Pavie 2010). Results are available within 15 to 30 minutes. Most frequently, rapid tests are based on immuno-chromatographic methods. Other techniques such as particle agglutination, ImmunoDOT and immunofiltration are also used (Giles 1999, Branson 2003, Greenwald 2006).
Almost all currently available rapid tests only detect HIV antibodies but not p24 antigen, corresponding to an (outdated) HIV test of the 3rd generation. Since 2009 a certified 4th generation rapid test (Determine HIV-1/2 Ag/Ab Combo, Inverness Medical) is available for the first time which not only detects but can also differentiate HIV antibodies and p24 antigen. However, in a comparative study the test exhibited deficiencies regarding the recognition of primary HIV infections. About one third of the samples of patients with acute HIV infection tested falsely negative. Reactivity was delayed by one week compared to a reference test (Mohrmann 2009).
Rapid tests are particularly suitable for use in emergency situations where the test result has immediate consequences. These include emergency operations and needlestick injuries. Also in pregnant women with unknown HIV status at delivery a rapid test can be useful. However, the cooperating laboratory should be contacted to indicate the need for a rapid HIV result. When necessary, the result of a conventional HIV test can be available within one hour upon receipt of the sample. Rapid tests are also useful in countries with poor medical infrastructure (WHO 1998, 2004) and in the context of low-threshold testing for individuals who would otherwise not be tested.
Rapid tests should be used only for initial orientation. The results of the testing should be confirmed at the earliest opportunity in a routine laboratory with a standard HIV test. Studies have shown that some rapid tests exhibit a lower sensitivity in comparison to conventional HIV tests. In one study in Cape Town, South Africa, a significant proportion of HIV-infected children had false-negative rapid test results (Claassen 2006).
The diagnostic window
The “diagnostic gap” or “window” indicates the time period between transmission of a pathogen and the onset of biochemical measurable infection markers such as antibodies, antigen or nucleic acids (Busch 1997).
At the earliest, HIV antibody production begins two weeks after transmission. HIV-specific antibodies can be detected after four weeks in 60-65% of cases, after six weeks in 80%, after eight weeks in 90% and after twelve weeks in 95% of cases. 4th generation diagnostic tests can shorten the diagnostic gap by simultaneous detection of p24 antigen (Gürtler 1998, Ly 2001). The p24 antigen is detectable about five days before seroconversion (the first occurrence of specific antibodies). The earliest lab marker is HIV RNA that is detectable approximately seven days before the p24 antigen (Fiebig 2003). In many cases HIV RNA can be detected by the second week after transmission. However, a negative result at this time point cannot exclude an infection.
A negative result in the HIV screening test precludes the existence of HIV antibodies and p24 antigen at the time of testing. The security of this result, however, depends particularly on the time interval from the possible transmission event. This has important consequences:
1. HIV testing immediately after a possible transmission is not meaningful, as no HIV antibodies are yet formed. An HIV test should therefore be carried out at the earliest in the 3rd week after exposure. Exception: If it needs to be documented for legal reasons (e.g., needle stick injury) that at the time of transmission no existing HIV infection was present.
2. An HIV infection can not be ruled out until three months after possible transmission with sufficient certainty. Check-ups should be performed two and six weeks and three months after exposure. A further test (after six months) is appropriate only in exceptional cases, for example, if there is suspicion of acute retroviral syndrome.
3. A negative test result is dependable only in the case of no re-exposure within the past three months (from the time of the original exposure).
HIV diagnostics in newborns
In newborns of HIV-infected mothers maternal antibodies may remain detectable until the age of 18 months (Moodley 1997, European collaborative study 1991). The antibodies are transplacentally transferred from the 32nd week of gestation although they do not have any protective effect. A serological HIV test for the detection or exclusion of vertical transmission of HIV is therefore not enough as a positive result will be expected in any case.
According to the German-Austrian recommendations for HIV therapy in pregnancy and in HIV-exposed infants (2008) at least two negative PCR results are required to exclude HIV transmission. The first HIV PCR should be performed after the first month of life (sensitivity 96%, specificity 99%), then again because of the nearly 100% sensitivity and specificity after the third month. Vertical transmission can be ruled out, however, only if there was no renewed risk of transmission in the meantime through breastfeeding.
Even with negative PCR results, the disappearance of maternal antibodies should be documented at least once. In the case of positive results, these must be confirmed by examination of a second sample.
HIV diagnostics after occupational exposure
After a needlestick injury or other occupational exposure a hepatitis B and C and HIV infection of the index patient should be excluded (of course, consent of the index patient is required). With regard to the potential necessary rapid start of post-exposure prophylaxis (PEP) a needle stick injury should always be considered an emergency. The earlier PEP is initiated, the better the chances of success (Puro 2004, Panlilio 2005). PEP should preferably be started within 24 hours of HIV exposure. If a rapid result of an HIV screening test is not available for logistical reasons, an HIV rapid test should be considered. To save time, PEP can be initiated immediately and terminated at any time in the case of a negative result.
If the index patient has no symptoms consistent with acute retroviral syndrome the negative result of the screening test excludes HIV infection with a high level of security. An HIV PCR test should be considered only if there is evidence of acute HIV infection of the index patient.
Conversely, if the index patient is infected with HIV or if the HIV status is unknown, HIV screening should be performed in the exposed person. For legal reasons, the first HIV test should take place immediately after the needlestick injury to document that no HIV infection was present at the moment of the accident. Check-ups should be carried out at 6 weeks, at 3 and at 6 months. If the index patient is infected with HIV, testing at 12 months is recommended (Ridzon 1997, Ciesielski 1997).
What is relevant in practice?
● The legal situation: Because of possible medical, social and legal consequences, an informed consent of the patient is required before performing an HIV test. Testing against the wishes of the patient is an invasion of privacy, potentially corresponding with legal consequences for the doctor. A written consent is not required, but the consent should be documented. In children or infants, the patient’s parents or legal guardians must agree. The special status of the HIV test is currently being debated. There is a demand for “destigmatization” and a “re-medicalization” of the HIV test (Manavi 2005, Beckwith 2005). The background is that the accompanying requirements of the informed consent are often discouraging, so it is often easier to omit doing the HIV test. Current CDC recommendations allow for the so-called “opt-out” in many situations: the patient is informed about the HIV test, but it will be performed without further detailed guidance, provided the patient does not explicitly reject testing (CDC 2006).
● Advice: There should not be any HIV testing without counseling and education. The patient should be informed about the testing algorithm and the possibilities and limitations of HIV testing. Particularly, the limitations of the (frequently demanded) HIV PCR in primary diagnostics should be addressed: while a sensitive method for detection, it is only conditionally suitable for the rapid exclusion of HIV infection or transmission. Due to the distress caused to the patient, the high cost of the PCR as a counter argument against the method is a rare deterrent for the patient. During the consult, all the possibilities of the test result and in particular the “diagnostic window” should be noted. A desired HIV test could also be an occasion to discuss the risk of transmission in general (also for other sexually transmitted diseases) and appropriate prevention methods with the patient.
● Reporting: A negative test result can possibly be reported by telephone if the patient has been previously advised of its value. The diagnosis of HIV, however, has to be given in a personal counseling interview by a physician (or expert virologist) only (in many places, the result can be given by a registered nurse or counselor). The response of a patient cannot be assessed adequately when reporting is done by telephone. Sometimes patients can develop suicidal thoughts. Similarly, the negative result of a confirmatory test following a reactive screening test should be personally discussed with regard to the possibility of an acute infection. Patients should be directed to an HIV-focused practice. In addition, the patient should be advised of regional counseling and care centers. The result of a reactive HIV screening test should never be reported before the result of the confirmatory test is available.
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