BLOOD DONATION
In blood donation an efficient national blood programme is an essential component of an effective health system. The critical requirement is access to safe and clinically effective blood and blood products for all patients requiring transfusion and their safe and appropriate use. Blood safety depends on the recruitment and retention of blood donors who are at low risk of transmitting infection, safe blood collection procedures, correct testing for transfusion-transmissible infections, blood grouping and compatibility testing and the appropriate use and safe administration of blood.
Apart from HIV infection, other diseases can also be transmitted through transfusions of blood or blood products. These include two other viral infections hepatitis B (HBV) and hepatitis C (HCV); syphilis; malaria, which is endemic in many tropical areas. While 80% of the world’s population lives in developing countries, people in developing countries are supported by only 20% of the worlds blood supply. In developing countries, most transfusions are given to: women, to treat haemorrhage as a complication of pregnancy; children with severe anaemia; and serious trauma victims. Each year, up to 4 million blood donations worldwide are not tested for HIV or HBV, very few donations are tested for HCV.
While in developed countries, whole blood donations are mainly from voluntary unpaid donors, in developing countries 80% of the donated blood comes from paid or replacement donors, who in general are more likely to carry transfusion transmissible infections (TTIs). As a result, the risk of disease from blood transfusions is much higher in developing countries.
Whole blood and blood products
Whole blood can be separated into blood products, which consist of plasma and cellular components (red cells, white cells and platelets). Plasma contains water, electrolytes, proteins and clotting (coagulation) factors. The latter being valuable for certain medical conditions such as haemophilia. Plasma needs to be frozen within 6.8 hours of the collection of whole blood and maintained, frozen solid, at a temperature of .20 oC or colder.
Whole blood and packed red cells must always be stored at a temperature of between +2 oC and +8 oC. If special anticoagulants are used, blood stored at this temperature can be kept for up to 35 days. Platelets must be kept at 20 oC to 2 oC, and stored no longer than 5 days. Plasma derivatives may be shared internationally, while whole blood and red cells, with a short shelf-life, are usually used nationally.
Screening of blood
The process of testing blood for transfusion-transmissible agents is known as screening Implicit in the practice of screening blood for infectious agents is the concept of good laboratory practice., to ensure correct blood grouping, compatibility of donor and recipient, and all processes leading to the provision of safe and effective blood and blood products.
In the case of HIV, several types of tests based on different technologies exist to detect HIV antibodies. Detailed information on the types of HIV tests and testing strategies is available in the UNAIDS Technical Update on HIV Testing Methods.
In general, ELISAs are more suitable for blood banks processing daily a large number of blood units, while simple and/or rapid tests are more appropriate for smaller blood banks with a limited number of donations each day. Several simple/rapid tests perform just as well as ELISAs and are also highly appropriate for use in emergency situations.
In blood donation to ensure safe blood the test selected for screening donated blood units should preferably be a combined HIV-1/HIV-2 test, which is highly sensitive. A test with a high sensitivity will not produce or will only rarely produce false-negative results, which is important for safeguarding the blood supply. HIV infection is most frequently diagnosed by detecting antibodies which the body produces as it tries to resist the virus. These antibodies usually begin to be produced within 3 to 8 weeks after the time of infection. The period following infection but before the antibodies become detectable is known as the window period. If a person donates blood during this window period, the usual antibody test may give a false-negative result even though the person is infected. Increasingly sensitive anti-HIV tests have shortened the window period to 21 days.
Tests also exist that detect the virus itself rather than antibodies to it; these are called HIV p24 antigen tests. With a test of this kind it is sometimes possible to detect HIV p24 antigen during the window period, if by coincidence the blood donor happens to be tested during the short peak of high levels of circulating virus particles.
Although in theory the HIV antigen test can shorten the window period by an additional
6 days, its use is of limited value and there still remains a window of one to two weeks. (See Bush & Alter, 1995 in the Key Materials.)
Several studies have shown that for minimizing the TTI risk, careful selection of donors is more efficient than HIV antigen testing. In addition, a wellfunctioning quality assurance programme will reduce the possibility of false-negative results resulting from technical errors. In most settings, HIV p24 antigen testing of the blood supply is not cost-effective and is not recommended by WHO.
Donated blood should be tested not only for HIV but also for syphilis, hepatitis B surface antigen, and if funds permit, hepatitis C antibodies. According to their geographic prevalence, screening for Chagas disease, HTLV-I/II and other TTIs may also need to be carried out. Some industrialized countries have suggested ending screening for syphilis because its prevalence in their donor populations is very low and because the agent does not survive if the blood is stored at between +4 oC and +8 oC for at least 72 hours. However, in many countries, blood is stored for only a short time before being transfused. In some of these countries the prevalence of syphilis in blood donors is high. Although syphilis is not a marker for HIV infection, it does indicate donors who have not deferred themselves, yet who are at risk of sexually transmitted diseases, including HIV. Thus the syphilis test serves as a marker of donor suitability.
Units of donated blood yielding reactive or indeterminate test results must be considered as probably infected and must be discarded according to universal safety instructions. If a blood donor is to be notified of a test result, reactive screening results must be confirmed.
Create a national blood transfusion service
Kenya has a national blood transfusion services, which means that the service makes all transfusion centers and blood banks part of a national network, accountable to the government or to a government appointed nonprofit organization. Such a service has been developed within the framework of the country’s health care infrastructure. Kenya national blood transfusion service has a national policy and plan for the NBTS, with the proper legislative and regulatory control and an adequate financial budget. For better service delivery the NBTS should be recognized as a clearly identified unit of the health care system (separate from general laboratory systems) and have its own budget and trained staff.
Important steps in setting up a NBTS include the following: obtain formal government commitment and support; develop a national blood policy and plan; identify an appropriate organization for the NBTS; appoint an executive committee and a chief medical officer for the NBTS, as well as where necessary an advisory committee of medical specialists; select and train staff with organizational, management, medical and technical skills; draw up a budget, and develop an appropriate financing system to make the blood programme sustainable; develop and implement a quality management system; develop and implement monitoring and evaluation systems for the service. The NBTS can be financed either through an annual government allocation or through a cost recovery fee. The cost-recovery fee is a charge for services to supply blood. It is paid by hospitals and other institutions using blood, and agreed upon in annual negotiations involving the government, hospital administrators and the blood transfusion service. The charge is intended to cover capital and recurrent cost items such as buildings, salaries and test kits. The blood or blood product itself should always ideally be free for those receiving transfusions, or else paid for through government allocations or a health insurance scheme.
Health workers must know that, good donor selection is an important part of the process of collecting safe blood. When donors present themselves at blood donation centres they need to be interviewed by trained staff, so that those who appear to have a high risk of being infected, or appear to be paid donors, are excluded. Potential donors whose poor health or nutritional status makes them unsuitable should also be excluded, for the sake of their own health as well as the health of the recipients.
In order to have safe blood, blood donors must be educated and counseled about the importance and responsibility of being a blood donor, such donors can make correct and informed decisions to donate, to self-exclude, or to self-defer. Self-exclusion means excluding themselves if they know or think that their blood may be unsafe as a result of risk behaviour, or because of the state of their own health. Self-deferral is postponing blood donation if there are temporary reasons for doing so. Some donors may be unwilling to self-exclude or self-defer even if they know that their blood may be unsafe. There may be peer group pressure on them to give blood, and they may not want others to know why they are unwilling to give blood. For this reason, it is important to give all donors an opportunity to tell counseling staff in the strictest confidence about their concerns.
In cases where the donor staff feels that a donor is unsuitable, they must have a mechanism to remove and dispose off the unit of blood after donation. This is called confidential unit exclusion (CUE). Educating the public, motivating and recruiting them, and retaining them, as donors who give blood regularly are all necessary. The ability to do these effectively depends on a well-staffed and well-funded blood donor recruitment unit, trained in dealing with mass media and in communication skills. Among other things, a blood donor recruitment team should do the following: write and produce educational materials for blood donors; plan and carry out educational campaigns in workplaces, schools and colleges to motivate, recruit and retain donors; set up a blood donor registry system; work out procedures for donor selection and deferral, as well as for donor notification; design mechanisms to retain donors (such as donor clubs); prepare guidelines and standard operating procedures, including procedures to ensure strict confidentiality for donors; train staff in counseling, techniques and the ability to select donors; set up links with other health facilities to refer donors, where necessary.
It is important that blood transfusion services move as rapidly as possible towards screening all blood. The tests should include at least HIV, HBV and syphilis and other TTIs as determined according to the prevalence and epidemiological risk. National guidelines should be developed and implemented for screening all blood donations using the most appropriate and effective testing strategies for each type of infection (see UNAIDS Technical Update, HIV Testing Methods). Items to be considered here include: the development of standard operating procedures and guidelines for screening, testing strategies, and a quality assurance programme; training of NBTS technical staff; the purchase, supply, storage and distribution of reagents and other materials used in testing, so as to ensure continuous testing.
Reduce unnecessary blood transfusions
It is important to minimize the number of inappropriate blood transfusions so as to reduce the risk of TTIs as well as other possible adverse reactions from transfusions. The following activities should be considered: developing a national standard operating procedure and national guidelines or indicators for giving transfusions; training people who prescribe blood to avoid unnecessary or inappropriate transfusions; ensuring accessibility and availability of blood substitutes for volume replacement, such as crystalloids and colloids, for use where appropriate; these will not transmit infections and can be obtained at a fraction of the cost of whole blood. Preventing the causes that lead to individuals requiring blood transfusions will not only save unnecessary transfusions and reduce transmission of TTIs, but will improve health in the long term, blood transfusions often given for chronic anemia are unnecessary if the underlying condition is treated and the patient given corrective therapy. Preventing diseases such as malaria and worm infestations, and raising health standards generally, are important measures to reduce unnecessary transfusions. Similarly, proper care for women before, during and after delivery, will greatly reduce blood loss and in turn reduce the need for transfusions.
The major focus of HIV prevention and control has been to promote the acceptance of risk-reducing behaviours, through prevention, counseling and testing, and to facilitate linkage to medical, prevention and other supports services. Testing has played a major role in reducing the transmission of HIV. Antibody testing to diagnose HIV was introduced in 1985. At that time, most available technologies employed a methodologic paradigm that made use of central facilities equipped with highly-trained technologists performing tests in batches. Such an approach allowed facilities to develop effective quality control techniques to ensure the reliable performance of tests, but also led to infrequent testing and long turn-around times.
The standard laboratory HIV testing protocol, which evolved in the 1990s, involved obtaining a blood specimen from the client and sending it to a licensed laboratory for testing. Most often, the central laboratory would perform an enzyme-linked immunoassay (EIA), in order to ensure that a reactive result was due to HIV exposure. A second, more specific assay, the Western blot, was widely used to confirm results. The patient would then need to return for a second visit to receive test results. Unfortunately, many patients would not return for their test results. The lag time between obtaining a specimen and providing results is a time of high anxiety and significant stress for many of these patients. While the time to perform an HIV antibody test is typically a few hours, the time required by the testing paradigm was typically two days to two weeks. Such long delays and the accompanying anxiety clearly contributed to the near 30% of patients who failed to return to counseling centres for their results.
The early and rapid diagnosis of HIV began to assume particular importance as effective combination antiretroviral therapy became available. Combination therapy contributes to reducing the risk of vertical and occupational HIV transmission while improving the quality of life and the longevity of people infected with HIV. A significant reduction in the lag time between risk exposure and the availability of testing results required the evolution of a new approach to HIV testing – the rapid HIV test. These tests are widely available internationally, including four that have been approved by the US Food and Drug Administration (FDA). Due to the fact that rapid, point-of-care testing offers the advantage that people do not need to return to obtain their test results, more people know their HIV status and if infected can be referred for treatment, prevention programs and social services more rapidly. People who know they are infected with HIV are more likely to practise risk-reduction, especially if a brief behavioural intervention is conducted at the patient visit on a donation day. Rapid testing offers the advantage of providing test results at the time of the behavioural intervention. Rapid diagnostic HIV testing has several clinical applications. This paper describes rapid testing and its role in reducing vertical HIV transmission for women who present in labour with unknown HIV status; reducing the risk of occupational transmission of HIV; and assisting in the diagnosis and counseling of patients with HIV. Rapid testing plays a crucial role in time-sensitive decisions regarding the need for prophylaxis to reduce transmission in cases of occupational exposures and women presenting in labour with unknown HIV status.6
R a p i d D i a g n o s t i c H I V T e s t s
Rapid tests to detect the HIV antibody are designed to allow healthcare providers to supply definitive negative and preliminary positive results in minutes at the time of an initial patient visit. In comparison, traditional enzyme immunoassays (EIAs) operate with a paradigm that requires specimen transmittal to a laboratory, the creation of batches of specimens for efficient, cost-effective processing, the use of expensive semi-automated or automated equipment and the presence of significant operator expertise to perform properly and reliably. These requirements often delay results from reaching the patient for as much as one to two weeks.7 Rapid HIV tests are comparable in sensitivity and specificity with traditional EIAs, but can be performed by testing personnel with limited technical expertise in as little as 10 minutes.
A number of HIV tests are being used throughout the world. In the US, four rapid tests have been approved by the FDA for commercial use: the Single Use Diagnostic System for HIV-1(SUDS, Abbott Laboratories, Abbott Park, IL –no longer marketed); OraQuick HIV-1 and the Oraquick Advance HIV-1/HIV-2 (Orasure Technologies, Bethlehem, PA); Reveal™ (MedMira Laboratories, Halifax, Nova Scotia); and Unigold Recombigen (Trinity Biotech plc. Wicklow, Ireland). Additional rapid tests are under consideration by the FDA. Many candidate rapid tests use a variety of specimen samples including serum, whole blood, plasma and/or oral mucosal transudate (OMT). Using whole blood, the four FDA-approved rapid tests have sensitivities ranging from 95.3% to 100% and specificities ranging from 96.7% to 100%. Performance results of six rapid tests – commercial tests using plasma as the test specimen demonstrate sensitivities ranging from 96.7% to 100% and specificities ranging from 98.5% to 100%.
The sensitivity and specificity of most rapid assays are comparable to those of non-rapid EIAs. In lowprevalence settings, the predictive value of a single rapid negative test result is very high. A negative rapid test does not, therefore, require further testing and negative results with result-specific counseling can be provided to most people at the time of their initial visit. Due to the fact that the positive predictive value varies with prevalence of HIV infection in the population tested, however, the positive predictive value will be low in populations with low prevalence.8 This phenomenon has led to a testing strategy requiring a reactive EIA or rapid test to be confirmed by a second, independent supplemental test.9 In studies conducted outside the US, specific combinations of two or more different rapid HIV assays have provided results as reliable as those from the EIA/Western blot combination, which is currently in widespread use.10 In the US, current recommendations require confirmatory testing to be conducted utilising a Western blot or an immunofluorescence assay (IFA).11
The ‘window’ of HIV diagnosis is dependent upon the diagnostic approach utilised to detect its presence. Following exposure, entry of the HIV virus into the bloodstream typically occurs between three and seven days later with detectable HIV-1 ribonucleic acid (RNA) being demonstrated between seven and 14 days later. A detectable p24 antigen may be present between 12 and 19 days, but antibody seroconversion and detection occurs between 30 and 60 days postexposure. The onset of symptoms typically occurs three to four weeks post-exposure and most patients are symptomatic with a flu-like illness at the time of antibody seroconversion.
The ease of performing some rapid tests led their manufacturers to seek and be granted waived test status under the federal Clinical Laboratory Improvement Amendments (CLIA). CLIA waived status allows testing facilities to offer HIV testing with less restrictive regulatory requirements. In order to ensure a highquality testing environment, however, the FDA has limited the test to registered laboratories and requires that the facility institute a quality assurance program. Guidelines from the US Centers for Disease Control and Prevention (CDC) recommend participation in a proficiency-testing program.7
R e c o m m e n d a t i o n s f o r R a p i d T e s t i n g o f Women in Labour
Prevention of vertical HIV transmission has been an important success story in the HIV pandemic. The risk of transmission has been reduced from approximately 25% to less than 2% by using currently recommended obstetrical interventions and pre-natal combination anti-retroviral therapy in women aware of their HIV infection early in pregnancy.6 Different state and local regulations specify policies and procedures related to HIV counseling and the testing of pregnant women.
Ideally, all pregnant women should be offered HIV testing during an initial pre-natal visit, to allow for timely initiation of treatment to reduce the chance of vertical transmission. A particular area of concern, however, is women who present in labour with unknown HIV status (HIV test results not documented on medical records). These women may not have been offered or opted for HIV counseling and testing during pregnancy or may not have received pre-natal care. Clinical trial data have shown that anti-retroviral medications, even when administration began during labour and delivery and continued in the neonatal period, can reduce mother-to-child HIV transmission by up to 50%.12–14
When women present in labour with unknown HIV status, the key to maximal peri-natal HIV risk reduction is rapid testing and initiation of short course therapy. The CDC-sponsored Mother–Infant Rapid Intervention at Delivery (MIRIAD) study showed that offering voluntary HIV testing during labour is feasible in obstetrical settings. In addition, point-of-care testing has been shown to provide results faster than sending specimens to the hospital laboratory for rapid HIV testing.15 The CDC recommends rapid HIV testing for women in labour whose HIV status is unknown. Women in labour who have a preliminary positive rapid test should be offered short-course therapy. One recommendation describes four options for short course therapy.12 Both the woman and the child should be referred for follow-up, preferably by providers with experience and expertise in treating HIV.
I n t e r p r e t a t i o n o f R a p i d T e s t R e s u l t s
Interpretation of rapid tests is the same as other HIV screening tests. A negative result from a single test is interpreted as being negative although, as with other HIV screening tests, if a person may have been exposed to HIV within three months of the test, a repeat test at a later time is recommended. A positive (or reactive) result is considered to be a preliminary positive test result. This must be confirmed using a Western blot or an IFA. This confirmatory testing should be done as soon as possible. If the rapid test is a preliminary positive and the confirmatory test is negative (discrepant results), both the rapid test and the confirmatory test should be repeated. A consultation with an infectious disease specialist is recommended. If the rapid test does not provide a valid test result, it is likely that the test kit did not work properly – in this case, the rapid test should be repeated.
C o u n s e l l i n g P a t i e n t s w i t h a N e g a t i v e R a p i d T e s t
Patients whose rapid HIV test result is negative can be told that they are not infected, unless they have had a recent (within three months) known, or possible, exposure to HIV. Retesting should be recommended for these patients, because sufficient time needs to elapse in order for the development of the antibodies (which are detected by the test) to progess.16,21
Confirmatory testing is always required to confirm a reactive rapid test result. The challenge is providing reactive (preliminary positive) results to patients without the benefit of a same-day confirmatory test. For all patients with a reactive rapid HIV test result, however, it is essential to: explain that this is a preliminary test and results need to be confirmed; emphasise the importance of confirmatory testing and schedule a return visit for the confirmatory test results; and underscore the importance of taking precautions to avoid the possibility of transmitting infection to others while awaiting results of confirmatory testing.21
C o n c l u s i o n
Rapid diagnostic HIV testing will improve the proportion of patients who receive their test results, help with clinical decision-making regarding the use of short course anti-retroviral therapy to reduce the risk of vertical HIV transmission for women who present in labour with unknown HIV status, and help determine the need for PEP for potential occupational exposures to HIV.16,17 As HIV counselling, testing and referrals advance, it is imperative that adjustments be made in recommendations and practices. People found to be infected with HIV should be referred for medical care by a provider with experience and expertise treating HIV disease and be referred for prevention services and social services. HIV/AIDS reporting requirements should be followed.
HIV Testing
Many persons with HIV do not get tested until late in their infection. Approximately 40% to 50% of patients with HIV infection are diagnosed with AIDS within 1 year of first testing HIV-positive [2,4–6]. Many persons who are tested do not return to learn their test results. The National Health Interview Survey found that 12.5% of persons tested in 1994 and 13.3% in 1995 did not receive their results [7], and the Centers for Disease Control and Prevention (CDC) estimates that in 2000, 31% of patients who tested HIV-positive at public-sector testing sites did not return to receive their results [8]. To reduce barriers to early diagnosis of HIV infection and increase access to treatment and prevention services, the CDC announced a new initiative, “Advancing HIV Prevention: New Strategies for a Changing Epidemic” (AHP) [8]. This multifaceted program stresses the importance of routinely offering HIV testing as part of the medical visit and expands on the 1993 recommendations for testing inpatients and outpatients in acute-care hospital settings [9].
Additionally, AHP stresses the importance of using rapid HIV tests to facilitate access to early diagnosis in high prevalence areas, for high-risk individuals, and for women during labor and delivery who have not previously been tested and in nontraditional testing settings. Rapid HIV tests can play an important role in HIV prevention activities and expand access to testing in both clinical and nonclinical settings. They can help overcome some of the barriers to early diagnosis and improve linkage to care of infected persons. This paper will review the operating and performance characteristics, quality assurance (QA) and laboratory requirements for currently available rapid HIV tests, and counseling implication.
Four rapid HIV tests have been approved by the US Food and Drug Administration (FDA): OraQuick® (and its newer version OraQuick® Advance) Rapid HIV-1/2 Antibody Test (OraSure Technologies, Inc., Bethlehem, PA); Reveal™ (and its newer version Reveal™ G2) Rapid HIV-1 Antibody Test (MedMira, Halifax, Nova Scotia); Uni-Gold Recombigen HIV Test (Trinity BioTech, Bray, Ireland); and Multispot HIV-1/HIV-2 Rapid Test (Bio-Rad Laboratories, Redmond, WA). Like conventional HIV enzyme immunoassays (EIAs), rapid HIV tests are screening tests that require confirmation if reactive. Though each of these rapid HIV tests has unique characteristics, they share many common features, including how the tests work, the use of external controls, and other requirements such as the product information sheets that are provided to patients.
OraQuick® Advance Rapid HIV-1/2 Antibody Test
On November 7, 2002, the FDA approved the OraQuick® Rapid HIV-1 Antibody Test for use on fingerstick blood samples. It received its CLIA waiver in January 2003. Subsequently, OraQuick® received approval for use with venipuncture whole blood and plasma (though OraQuick® used with plasma is classified as moderate complexity under CLIA). In 2004, OraQuick® Advance received FDA approval for use with oral fluid and for detection of both HIV-1 and HIV-2. The OraQuick® test device is shown in Figure 1. The paddle-shaped device contains a nitrocellulose strip, upon which a stripe of synthetic gp41 peptides represent- ing the HIV-1 envelope and the gp36 region of the HIV-2 envelope have been applied in the “T” (test) location, and a stripe of goat antihuman IgG in the “C” (control) location. The specimen of blood or plasma is added directly to the developer vial. For oral fluid testing, the oral fluid sample is collected by swabbing the gums with the paddle-shaped device. The test device is then added to the developer vial. If HIV antibodies are present in the specimen, they bind to the peptides causing a red line to appear in the test location. As the solution migrates further, it encounters the antihuman IgG control, and if an adequate specimen was added, a red line appears in the control location.
The test result should be read no sooner than 20 minutes and no later than 40 minutes after the test device is inserted into the developer vial. A red line at both the test and control location indicates a valid reactive test result; a red line only in the control location indicates a valid negative test result. The test is invalid and should be repeated with a new device if no line appears at the control location or if lines appear outside the areas indicated by the triangles [10].
Designed as a point-of-care HIV test, OraQuick® has been used in numerous settings including labor and delivery [11•], ambulatory clinical sites [12], emergency departments [13,14], hospital inpatient services [15] (Greenwald JL, unpublished data), correctional facilities [16], and for occupational exposures [17–19]. Additionally, OraQuick® has also been used by the military in battlefield operations [20].
Reveal™ G2 Rapid HIV-1 Antibody Test
On April 17, 2003, the FDA approved the Reveal™ Rapid HIV-1 Antibody Test to detect HIV antibodies in serum or plasma. In June 2004, it was superseded by the second generation Reveal™ G2 test, which incorporates an internal control [21]. Reveal™ G2 consists of a test cartridge and a proprietary colorimetric detection agent. Positive and negative external controls, which must also be reconstituted, are supplied with the kit. Reveal™ is considered reactive if both the red control line and central red test dot appear, negative if only the control line appears, and invalid if the control line does not appear (Fig. 2). The Reveal™ G2 only takes 3 minutes to run [22]. However because it requires serum or plasma from centrifuged blood samples and several reagent steps, it is classified as a moderate complexity test under CLIA and is usually performed in a clinical laboratory.
Uni-Gold Recombigen® HIV Test
The Uni-Gold Recombigen® HIV Test received FDA approval in December 2003 for testing whole blood, serum, and plasma for antibodies to HIV-1. It was waived under CLIA in 2004 for use with venipuncture and fingerstick whole blood specimens [23]. The device consists of a rectangular plastic test cartridge and a dropper bottle of buffer solution (Fig. 3). Peptides from the immunodominant region of the HIV-1 envelope are immobilized on a nitrocellulose strip in the test region. Reagents are also bound at the control region to indicate whether the test is functioning correctly, but these do not detect IgG and thus appearance of the control line does not validate that adequate patient specimen has been added. One drop of specimen is added to the specimen well on the test cartridge followed by four drops of wash buffer. The specimen combines with the colorimetric reagent and migrates along the nitrocellulose strip past the test and control regions. The test is read 10 to 12 minutes after specimen is added. A line in both the test and control regions indicates a reactive test; a line in only the control region indicates a negative test. When used with whole blood, the test is valid only if the control line is present and the sample well is red, indicating that an adequate blood sample has been added [24].
Multispot HIV-1/HIV-2 Rapid Test
The Multispot HIV-1/HIV-2 Rapid Test received FDA approval in November, 2004 [25]. Multispot is classified as a moderate complexity under CLIA, approved for use on fresh or frozen serum and plasma to both detect and distinguish HIV-1 from HIV-2. Multispot consists of a test cartridge and five reagents: specimen diluent, wash solution, conjugate, development reagent, and stop solution. The cartridge contains a membrane on which microparticles have been immobilized in four spots. Two of the spots consist of ecombinant and synthetic gp41 peptides to detect HIV-1 antibodies; one consists of synthetic gp36 peptides to detect antibodies to HIV-2; and the fourth spot consists of goat antihuman IgG as the internal control.
The test is considered positive for HIV-1 if the control spot and either or both of the HIV-1 spots turn purple, and positive for HIV-2 if the control and HIV-2 spots appear (Fig. 4). If purple appears in the control spot, the HIV-2 spot, and one or both of the HIV-1 spots, the test is considered HIV reactive (undifferentiated). In this case, the specimen may be tested by additional methods, which allow differentiation between HIV-1 and HIV-2. The test is negative when only the control spot appears. The absence of the control spot indicates an invalid result, regardless of any other spot pattern.
Rapid HIV Antibody Test Performance and Interpretation of Test Results
Like conventional EIAs, rapid HIV tests are screening tests. If performed correctly, they detect HIV antibodies with sensitivities similar to currently available EIAs [10,22,24–29] (Table 1). A negative rapid HIV test result requires no further confirmatory testing. False negative results, though rare, may occur in a person who has been acutely infected but who has not yet developed HIV antibodies. Therefore, any patient testing negative who
has had known or suspected exposure to HIV within 3 months should be instructed to retest 3 months after the exposure date [30]. Additionally, false-negative rapid HIV
test results have been observed in some patients receiving highly active antiretroviral therapy with undetectable virus in whom levels of HIV antibody have waned below the level of detection by the rapid HIV test [31].
A reactive result from any of the four rapid HIV tests is interpreted as a “preliminary positive” and requires confirmation by a more specific assay, typically a Western Blot (WB) or immunofluorescent assay (IFA) [10,22,24,25]. Performing a standard EIA screening prior to confirmatory testing is not required. However, if an EIA is performed, the specimen must still proceed to WB or IFA testing regardless of the EIA result. A positive WB or IFA confirms the diagnosis of HIV infection. If the con-firmatory test yields negative or indeterminate results, follow-up HIV testing should be performed on a blood specimen collected 4 weeks after the initial reactive rapid HIV test result [32•] as some patients newly infected with HIV may not have developed antibody levels sufficient to produce a positive WB or IFA [33].
Table 1 presents the test performances of US FDAapproved rapid HIV tests. It is important to note that because the test specificities are less than 100%, false positive rapid test results are an expected but rare event.
When testing low seroprevalence areas, a higher proportion of reactive tests will be false positives because there are few true positives in low-prevalence populations. The causes of falsely positive rapid HIV tests (ie, a reactive rapid HIV test with a negative or indeterminate confirmatory test) are poorly understood. Certain medical conditions may be associated with a slightly increased risk for false-positive OraQuick® rapid HIV tests (eg, hepatitis
A and B viruses, Epstein-Barr virus, multiparity, and the serologic presence of rheumatoid factor) [10].
Quality Assurance for CLIA-waived Rapid HIV Antibody Tests
Although CLIA-waived rapid HIV test devices are easy to use and can provide reliable results when the manufacturer’s directions are followed, mistakes can occur at any point in the testing process, including storage and testing area temperature, test kit shelf-life, specimen collection, test performance and results interpretation, referring specimens for confirmatory testing, managing confirmatory test results, etc. To reduce mistakes and to ensure that the FDA restrictions for sale of the test are followed, a site that performs rapid HIV tests must have a QA program in place before offering these tests.
In January 2003, the CDC convened a panel of experts including laboratory scientists and individuals from the FDA and the Centers for Medicare and Medicaid
Services to develop guidelines that outline the basic parts of a rapid HIV test QA program [32•]. The Quality Figure 3. Uni-Gold Recombigen® HIV test. A Rapid Review of Rapid HIV Antibody Tests Greenwald et al. 129 Assurance Guidelines for Testing Using the OraQuick® Rapid
HIV-1 Antibody Test are intended to assist a range of providers in developing policies, processes and procedures to ensure high quality HIV testing services. These guidelines include 1) the basics of a QA program for testing using OraQuick®, 2) an overview of government rules that apply to using this test, and 3) examples of forms/checklists that can be used to keep track of QA outcomes.
Counseling with Rapid HIV Antibody Tests
Counseling for patients choosing rapid HIV testing involves some differences compared with conventional testing, including assessing preparedness for clients to receive test results in the same session and explaining the meaning of preliminary positive results. Information can be provided either face-to-face or in a pamphlet, brochure, or video [34].
Patients with reactive rapid test results must be counseled in simple terms about the meaning of a reactive test. The provider must emphasize the need for a confirmatory test and schedule a return visit for results. Providers offering rapid HIV testing should be able to collect blood or oral fluid specimens on-site for confirmatory testing. All patients with reactive tests should be counseled on risk-reduction behaviors while awaiting the results of confirmatory testing. A simple message to convey this information could be “Your preliminary test result is positive, but we won’t know for sure if you are infected with HIV until we get the results from your confirmatory test. In the meantime, you should take precautions to avoid transmitting the virus” [34]. The New York State Department of Health AIDS Institute has also created guidelines for how to discuss reactive results stratifying the language based on the patient’s level of risk for HIV infection. For clients at high risk, the guidelines suggest saying “Based on your risk factors, it is highly likely that the preliminary test result is correct and that you have HIV” (emphasis added). For those at low risk, the phrase “quite likely” is recommended, and for those with no admitted risk factors, they advise informing them “There is a chance that this result could be a false positive” [35].
Physicians and counseling staff may be apprehensive about rapid testing specifically with regards to the ability to handle preliminary positive test results at any time. Data from RESPECT-2, a large, randomized, controlled trial that compared different forms of HIV testing and risk-reduction counseling in clients at sexually transmitted disease (STD) clinics in the United States, found that after gaining experience in the field, the majority of counselors preferred rapid testing, felt that rapid HIV testing sessions resulted in enhanced counseling, and felt that it was more convenient for both clients and counseling staff [36]. Although some have expressed concern about how counselors and clients will deal with discussing and understanding reactive results [37], others have noted that providers have extensive experience managing preliminary positive test results (eg, abnormal mammograms that require biopsies and abnormal pap smears that require colposcopy) [38] and studies of rapid testing have demonstrated good client understanding of results [39].
Providing HIV counseling and testing may be challenging in some health care settings. Because the average primary care office visit in the United States is less than 18 minutes long [40], even the “brief” counseling protocol of RESPECT-2 could take up an entire office visit. In these situations, alternative procedures for HIV counseling with rapid testing should be considered, eg, providing information either in a face-to-face meeting with a counselor or in a pamphlet, brochure, or video [34].
Apart from HIV infection, other diseases can also be transmitted through transfusions of blood or blood products. These include two other viral infections hepatitis B (HBV) and hepatitis C (HCV); syphilis; malaria, which is endemic in many tropical areas. While 80% of the world’s population lives in developing countries, people in developing countries are supported by only 20% of the worlds blood supply. In developing countries, most transfusions are given to: women, to treat haemorrhage as a complication of pregnancy; children with severe anaemia; and serious trauma victims. Each year, up to 4 million blood donations worldwide are not tested for HIV or HBV, very few donations are tested for HCV.
While in developed countries, whole blood donations are mainly from voluntary unpaid donors, in developing countries 80% of the donated blood comes from paid or replacement donors, who in general are more likely to carry transfusion transmissible infections (TTIs). As a result, the risk of disease from blood transfusions is much higher in developing countries.
Whole blood and blood products
Whole blood can be separated into blood products, which consist of plasma and cellular components (red cells, white cells and platelets). Plasma contains water, electrolytes, proteins and clotting (coagulation) factors. The latter being valuable for certain medical conditions such as haemophilia. Plasma needs to be frozen within 6.8 hours of the collection of whole blood and maintained, frozen solid, at a temperature of .20 oC or colder.
Whole blood and packed red cells must always be stored at a temperature of between +2 oC and +8 oC. If special anticoagulants are used, blood stored at this temperature can be kept for up to 35 days. Platelets must be kept at 20 oC to 2 oC, and stored no longer than 5 days. Plasma derivatives may be shared internationally, while whole blood and red cells, with a short shelf-life, are usually used nationally.
Screening of blood
The process of testing blood for transfusion-transmissible agents is known as screening Implicit in the practice of screening blood for infectious agents is the concept of good laboratory practice., to ensure correct blood grouping, compatibility of donor and recipient, and all processes leading to the provision of safe and effective blood and blood products.
In the case of HIV, several types of tests based on different technologies exist to detect HIV antibodies. Detailed information on the types of HIV tests and testing strategies is available in the UNAIDS Technical Update on HIV Testing Methods.
In general, ELISAs are more suitable for blood banks processing daily a large number of blood units, while simple and/or rapid tests are more appropriate for smaller blood banks with a limited number of donations each day. Several simple/rapid tests perform just as well as ELISAs and are also highly appropriate for use in emergency situations.
In blood donation to ensure safe blood the test selected for screening donated blood units should preferably be a combined HIV-1/HIV-2 test, which is highly sensitive. A test with a high sensitivity will not produce or will only rarely produce false-negative results, which is important for safeguarding the blood supply. HIV infection is most frequently diagnosed by detecting antibodies which the body produces as it tries to resist the virus. These antibodies usually begin to be produced within 3 to 8 weeks after the time of infection. The period following infection but before the antibodies become detectable is known as the window period. If a person donates blood during this window period, the usual antibody test may give a false-negative result even though the person is infected. Increasingly sensitive anti-HIV tests have shortened the window period to 21 days.
Tests also exist that detect the virus itself rather than antibodies to it; these are called HIV p24 antigen tests. With a test of this kind it is sometimes possible to detect HIV p24 antigen during the window period, if by coincidence the blood donor happens to be tested during the short peak of high levels of circulating virus particles.
Although in theory the HIV antigen test can shorten the window period by an additional
6 days, its use is of limited value and there still remains a window of one to two weeks. (See Bush & Alter, 1995 in the Key Materials.)
Several studies have shown that for minimizing the TTI risk, careful selection of donors is more efficient than HIV antigen testing. In addition, a wellfunctioning quality assurance programme will reduce the possibility of false-negative results resulting from technical errors. In most settings, HIV p24 antigen testing of the blood supply is not cost-effective and is not recommended by WHO.
Donated blood should be tested not only for HIV but also for syphilis, hepatitis B surface antigen, and if funds permit, hepatitis C antibodies. According to their geographic prevalence, screening for Chagas disease, HTLV-I/II and other TTIs may also need to be carried out. Some industrialized countries have suggested ending screening for syphilis because its prevalence in their donor populations is very low and because the agent does not survive if the blood is stored at between +4 oC and +8 oC for at least 72 hours. However, in many countries, blood is stored for only a short time before being transfused. In some of these countries the prevalence of syphilis in blood donors is high. Although syphilis is not a marker for HIV infection, it does indicate donors who have not deferred themselves, yet who are at risk of sexually transmitted diseases, including HIV. Thus the syphilis test serves as a marker of donor suitability.
Units of donated blood yielding reactive or indeterminate test results must be considered as probably infected and must be discarded according to universal safety instructions. If a blood donor is to be notified of a test result, reactive screening results must be confirmed.
Create a national blood transfusion service
Kenya has a national blood transfusion services, which means that the service makes all transfusion centers and blood banks part of a national network, accountable to the government or to a government appointed nonprofit organization. Such a service has been developed within the framework of the country’s health care infrastructure. Kenya national blood transfusion service has a national policy and plan for the NBTS, with the proper legislative and regulatory control and an adequate financial budget. For better service delivery the NBTS should be recognized as a clearly identified unit of the health care system (separate from general laboratory systems) and have its own budget and trained staff.
Important steps in setting up a NBTS include the following: obtain formal government commitment and support; develop a national blood policy and plan; identify an appropriate organization for the NBTS; appoint an executive committee and a chief medical officer for the NBTS, as well as where necessary an advisory committee of medical specialists; select and train staff with organizational, management, medical and technical skills; draw up a budget, and develop an appropriate financing system to make the blood programme sustainable; develop and implement a quality management system; develop and implement monitoring and evaluation systems for the service. The NBTS can be financed either through an annual government allocation or through a cost recovery fee. The cost-recovery fee is a charge for services to supply blood. It is paid by hospitals and other institutions using blood, and agreed upon in annual negotiations involving the government, hospital administrators and the blood transfusion service. The charge is intended to cover capital and recurrent cost items such as buildings, salaries and test kits. The blood or blood product itself should always ideally be free for those receiving transfusions, or else paid for through government allocations or a health insurance scheme.
Health workers must know that, good donor selection is an important part of the process of collecting safe blood. When donors present themselves at blood donation centres they need to be interviewed by trained staff, so that those who appear to have a high risk of being infected, or appear to be paid donors, are excluded. Potential donors whose poor health or nutritional status makes them unsuitable should also be excluded, for the sake of their own health as well as the health of the recipients.
In order to have safe blood, blood donors must be educated and counseled about the importance and responsibility of being a blood donor, such donors can make correct and informed decisions to donate, to self-exclude, or to self-defer. Self-exclusion means excluding themselves if they know or think that their blood may be unsafe as a result of risk behaviour, or because of the state of their own health. Self-deferral is postponing blood donation if there are temporary reasons for doing so. Some donors may be unwilling to self-exclude or self-defer even if they know that their blood may be unsafe. There may be peer group pressure on them to give blood, and they may not want others to know why they are unwilling to give blood. For this reason, it is important to give all donors an opportunity to tell counseling staff in the strictest confidence about their concerns.
In cases where the donor staff feels that a donor is unsuitable, they must have a mechanism to remove and dispose off the unit of blood after donation. This is called confidential unit exclusion (CUE). Educating the public, motivating and recruiting them, and retaining them, as donors who give blood regularly are all necessary. The ability to do these effectively depends on a well-staffed and well-funded blood donor recruitment unit, trained in dealing with mass media and in communication skills. Among other things, a blood donor recruitment team should do the following: write and produce educational materials for blood donors; plan and carry out educational campaigns in workplaces, schools and colleges to motivate, recruit and retain donors; set up a blood donor registry system; work out procedures for donor selection and deferral, as well as for donor notification; design mechanisms to retain donors (such as donor clubs); prepare guidelines and standard operating procedures, including procedures to ensure strict confidentiality for donors; train staff in counseling, techniques and the ability to select donors; set up links with other health facilities to refer donors, where necessary.
It is important that blood transfusion services move as rapidly as possible towards screening all blood. The tests should include at least HIV, HBV and syphilis and other TTIs as determined according to the prevalence and epidemiological risk. National guidelines should be developed and implemented for screening all blood donations using the most appropriate and effective testing strategies for each type of infection (see UNAIDS Technical Update, HIV Testing Methods). Items to be considered here include: the development of standard operating procedures and guidelines for screening, testing strategies, and a quality assurance programme; training of NBTS technical staff; the purchase, supply, storage and distribution of reagents and other materials used in testing, so as to ensure continuous testing.
Reduce unnecessary blood transfusions
It is important to minimize the number of inappropriate blood transfusions so as to reduce the risk of TTIs as well as other possible adverse reactions from transfusions. The following activities should be considered: developing a national standard operating procedure and national guidelines or indicators for giving transfusions; training people who prescribe blood to avoid unnecessary or inappropriate transfusions; ensuring accessibility and availability of blood substitutes for volume replacement, such as crystalloids and colloids, for use where appropriate; these will not transmit infections and can be obtained at a fraction of the cost of whole blood. Preventing the causes that lead to individuals requiring blood transfusions will not only save unnecessary transfusions and reduce transmission of TTIs, but will improve health in the long term, blood transfusions often given for chronic anemia are unnecessary if the underlying condition is treated and the patient given corrective therapy. Preventing diseases such as malaria and worm infestations, and raising health standards generally, are important measures to reduce unnecessary transfusions. Similarly, proper care for women before, during and after delivery, will greatly reduce blood loss and in turn reduce the need for transfusions.
The major focus of HIV prevention and control has been to promote the acceptance of risk-reducing behaviours, through prevention, counseling and testing, and to facilitate linkage to medical, prevention and other supports services. Testing has played a major role in reducing the transmission of HIV. Antibody testing to diagnose HIV was introduced in 1985. At that time, most available technologies employed a methodologic paradigm that made use of central facilities equipped with highly-trained technologists performing tests in batches. Such an approach allowed facilities to develop effective quality control techniques to ensure the reliable performance of tests, but also led to infrequent testing and long turn-around times.
The standard laboratory HIV testing protocol, which evolved in the 1990s, involved obtaining a blood specimen from the client and sending it to a licensed laboratory for testing. Most often, the central laboratory would perform an enzyme-linked immunoassay (EIA), in order to ensure that a reactive result was due to HIV exposure. A second, more specific assay, the Western blot, was widely used to confirm results. The patient would then need to return for a second visit to receive test results. Unfortunately, many patients would not return for their test results. The lag time between obtaining a specimen and providing results is a time of high anxiety and significant stress for many of these patients. While the time to perform an HIV antibody test is typically a few hours, the time required by the testing paradigm was typically two days to two weeks. Such long delays and the accompanying anxiety clearly contributed to the near 30% of patients who failed to return to counseling centres for their results.
The early and rapid diagnosis of HIV began to assume particular importance as effective combination antiretroviral therapy became available. Combination therapy contributes to reducing the risk of vertical and occupational HIV transmission while improving the quality of life and the longevity of people infected with HIV. A significant reduction in the lag time between risk exposure and the availability of testing results required the evolution of a new approach to HIV testing – the rapid HIV test. These tests are widely available internationally, including four that have been approved by the US Food and Drug Administration (FDA). Due to the fact that rapid, point-of-care testing offers the advantage that people do not need to return to obtain their test results, more people know their HIV status and if infected can be referred for treatment, prevention programs and social services more rapidly. People who know they are infected with HIV are more likely to practise risk-reduction, especially if a brief behavioural intervention is conducted at the patient visit on a donation day. Rapid testing offers the advantage of providing test results at the time of the behavioural intervention. Rapid diagnostic HIV testing has several clinical applications. This paper describes rapid testing and its role in reducing vertical HIV transmission for women who present in labour with unknown HIV status; reducing the risk of occupational transmission of HIV; and assisting in the diagnosis and counseling of patients with HIV. Rapid testing plays a crucial role in time-sensitive decisions regarding the need for prophylaxis to reduce transmission in cases of occupational exposures and women presenting in labour with unknown HIV status.6
R a p i d D i a g n o s t i c H I V T e s t s
Rapid tests to detect the HIV antibody are designed to allow healthcare providers to supply definitive negative and preliminary positive results in minutes at the time of an initial patient visit. In comparison, traditional enzyme immunoassays (EIAs) operate with a paradigm that requires specimen transmittal to a laboratory, the creation of batches of specimens for efficient, cost-effective processing, the use of expensive semi-automated or automated equipment and the presence of significant operator expertise to perform properly and reliably. These requirements often delay results from reaching the patient for as much as one to two weeks.7 Rapid HIV tests are comparable in sensitivity and specificity with traditional EIAs, but can be performed by testing personnel with limited technical expertise in as little as 10 minutes.
A number of HIV tests are being used throughout the world. In the US, four rapid tests have been approved by the FDA for commercial use: the Single Use Diagnostic System for HIV-1(SUDS, Abbott Laboratories, Abbott Park, IL –no longer marketed); OraQuick HIV-1 and the Oraquick Advance HIV-1/HIV-2 (Orasure Technologies, Bethlehem, PA); Reveal™ (MedMira Laboratories, Halifax, Nova Scotia); and Unigold Recombigen (Trinity Biotech plc. Wicklow, Ireland). Additional rapid tests are under consideration by the FDA. Many candidate rapid tests use a variety of specimen samples including serum, whole blood, plasma and/or oral mucosal transudate (OMT). Using whole blood, the four FDA-approved rapid tests have sensitivities ranging from 95.3% to 100% and specificities ranging from 96.7% to 100%. Performance results of six rapid tests – commercial tests using plasma as the test specimen demonstrate sensitivities ranging from 96.7% to 100% and specificities ranging from 98.5% to 100%.
The sensitivity and specificity of most rapid assays are comparable to those of non-rapid EIAs. In lowprevalence settings, the predictive value of a single rapid negative test result is very high. A negative rapid test does not, therefore, require further testing and negative results with result-specific counseling can be provided to most people at the time of their initial visit. Due to the fact that the positive predictive value varies with prevalence of HIV infection in the population tested, however, the positive predictive value will be low in populations with low prevalence.8 This phenomenon has led to a testing strategy requiring a reactive EIA or rapid test to be confirmed by a second, independent supplemental test.9 In studies conducted outside the US, specific combinations of two or more different rapid HIV assays have provided results as reliable as those from the EIA/Western blot combination, which is currently in widespread use.10 In the US, current recommendations require confirmatory testing to be conducted utilising a Western blot or an immunofluorescence assay (IFA).11
The ‘window’ of HIV diagnosis is dependent upon the diagnostic approach utilised to detect its presence. Following exposure, entry of the HIV virus into the bloodstream typically occurs between three and seven days later with detectable HIV-1 ribonucleic acid (RNA) being demonstrated between seven and 14 days later. A detectable p24 antigen may be present between 12 and 19 days, but antibody seroconversion and detection occurs between 30 and 60 days postexposure. The onset of symptoms typically occurs three to four weeks post-exposure and most patients are symptomatic with a flu-like illness at the time of antibody seroconversion.
The ease of performing some rapid tests led their manufacturers to seek and be granted waived test status under the federal Clinical Laboratory Improvement Amendments (CLIA). CLIA waived status allows testing facilities to offer HIV testing with less restrictive regulatory requirements. In order to ensure a highquality testing environment, however, the FDA has limited the test to registered laboratories and requires that the facility institute a quality assurance program. Guidelines from the US Centers for Disease Control and Prevention (CDC) recommend participation in a proficiency-testing program.7
R e c o m m e n d a t i o n s f o r R a p i d T e s t i n g o f Women in Labour
Prevention of vertical HIV transmission has been an important success story in the HIV pandemic. The risk of transmission has been reduced from approximately 25% to less than 2% by using currently recommended obstetrical interventions and pre-natal combination anti-retroviral therapy in women aware of their HIV infection early in pregnancy.6 Different state and local regulations specify policies and procedures related to HIV counseling and the testing of pregnant women.
Ideally, all pregnant women should be offered HIV testing during an initial pre-natal visit, to allow for timely initiation of treatment to reduce the chance of vertical transmission. A particular area of concern, however, is women who present in labour with unknown HIV status (HIV test results not documented on medical records). These women may not have been offered or opted for HIV counseling and testing during pregnancy or may not have received pre-natal care. Clinical trial data have shown that anti-retroviral medications, even when administration began during labour and delivery and continued in the neonatal period, can reduce mother-to-child HIV transmission by up to 50%.12–14
When women present in labour with unknown HIV status, the key to maximal peri-natal HIV risk reduction is rapid testing and initiation of short course therapy. The CDC-sponsored Mother–Infant Rapid Intervention at Delivery (MIRIAD) study showed that offering voluntary HIV testing during labour is feasible in obstetrical settings. In addition, point-of-care testing has been shown to provide results faster than sending specimens to the hospital laboratory for rapid HIV testing.15 The CDC recommends rapid HIV testing for women in labour whose HIV status is unknown. Women in labour who have a preliminary positive rapid test should be offered short-course therapy. One recommendation describes four options for short course therapy.12 Both the woman and the child should be referred for follow-up, preferably by providers with experience and expertise in treating HIV.
I n t e r p r e t a t i o n o f R a p i d T e s t R e s u l t s
Interpretation of rapid tests is the same as other HIV screening tests. A negative result from a single test is interpreted as being negative although, as with other HIV screening tests, if a person may have been exposed to HIV within three months of the test, a repeat test at a later time is recommended. A positive (or reactive) result is considered to be a preliminary positive test result. This must be confirmed using a Western blot or an IFA. This confirmatory testing should be done as soon as possible. If the rapid test is a preliminary positive and the confirmatory test is negative (discrepant results), both the rapid test and the confirmatory test should be repeated. A consultation with an infectious disease specialist is recommended. If the rapid test does not provide a valid test result, it is likely that the test kit did not work properly – in this case, the rapid test should be repeated.
C o u n s e l l i n g P a t i e n t s w i t h a N e g a t i v e R a p i d T e s t
Patients whose rapid HIV test result is negative can be told that they are not infected, unless they have had a recent (within three months) known, or possible, exposure to HIV. Retesting should be recommended for these patients, because sufficient time needs to elapse in order for the development of the antibodies (which are detected by the test) to progess.16,21
Confirmatory testing is always required to confirm a reactive rapid test result. The challenge is providing reactive (preliminary positive) results to patients without the benefit of a same-day confirmatory test. For all patients with a reactive rapid HIV test result, however, it is essential to: explain that this is a preliminary test and results need to be confirmed; emphasise the importance of confirmatory testing and schedule a return visit for the confirmatory test results; and underscore the importance of taking precautions to avoid the possibility of transmitting infection to others while awaiting results of confirmatory testing.21
C o n c l u s i o n
Rapid diagnostic HIV testing will improve the proportion of patients who receive their test results, help with clinical decision-making regarding the use of short course anti-retroviral therapy to reduce the risk of vertical HIV transmission for women who present in labour with unknown HIV status, and help determine the need for PEP for potential occupational exposures to HIV.16,17 As HIV counselling, testing and referrals advance, it is imperative that adjustments be made in recommendations and practices. People found to be infected with HIV should be referred for medical care by a provider with experience and expertise treating HIV disease and be referred for prevention services and social services. HIV/AIDS reporting requirements should be followed.
HIV Testing
Many persons with HIV do not get tested until late in their infection. Approximately 40% to 50% of patients with HIV infection are diagnosed with AIDS within 1 year of first testing HIV-positive [2,4–6]. Many persons who are tested do not return to learn their test results. The National Health Interview Survey found that 12.5% of persons tested in 1994 and 13.3% in 1995 did not receive their results [7], and the Centers for Disease Control and Prevention (CDC) estimates that in 2000, 31% of patients who tested HIV-positive at public-sector testing sites did not return to receive their results [8]. To reduce barriers to early diagnosis of HIV infection and increase access to treatment and prevention services, the CDC announced a new initiative, “Advancing HIV Prevention: New Strategies for a Changing Epidemic” (AHP) [8]. This multifaceted program stresses the importance of routinely offering HIV testing as part of the medical visit and expands on the 1993 recommendations for testing inpatients and outpatients in acute-care hospital settings [9].
Additionally, AHP stresses the importance of using rapid HIV tests to facilitate access to early diagnosis in high prevalence areas, for high-risk individuals, and for women during labor and delivery who have not previously been tested and in nontraditional testing settings. Rapid HIV tests can play an important role in HIV prevention activities and expand access to testing in both clinical and nonclinical settings. They can help overcome some of the barriers to early diagnosis and improve linkage to care of infected persons. This paper will review the operating and performance characteristics, quality assurance (QA) and laboratory requirements for currently available rapid HIV tests, and counseling implication.
Four rapid HIV tests have been approved by the US Food and Drug Administration (FDA): OraQuick® (and its newer version OraQuick® Advance) Rapid HIV-1/2 Antibody Test (OraSure Technologies, Inc., Bethlehem, PA); Reveal™ (and its newer version Reveal™ G2) Rapid HIV-1 Antibody Test (MedMira, Halifax, Nova Scotia); Uni-Gold Recombigen HIV Test (Trinity BioTech, Bray, Ireland); and Multispot HIV-1/HIV-2 Rapid Test (Bio-Rad Laboratories, Redmond, WA). Like conventional HIV enzyme immunoassays (EIAs), rapid HIV tests are screening tests that require confirmation if reactive. Though each of these rapid HIV tests has unique characteristics, they share many common features, including how the tests work, the use of external controls, and other requirements such as the product information sheets that are provided to patients.
OraQuick® Advance Rapid HIV-1/2 Antibody Test
On November 7, 2002, the FDA approved the OraQuick® Rapid HIV-1 Antibody Test for use on fingerstick blood samples. It received its CLIA waiver in January 2003. Subsequently, OraQuick® received approval for use with venipuncture whole blood and plasma (though OraQuick® used with plasma is classified as moderate complexity under CLIA). In 2004, OraQuick® Advance received FDA approval for use with oral fluid and for detection of both HIV-1 and HIV-2. The OraQuick® test device is shown in Figure 1. The paddle-shaped device contains a nitrocellulose strip, upon which a stripe of synthetic gp41 peptides represent- ing the HIV-1 envelope and the gp36 region of the HIV-2 envelope have been applied in the “T” (test) location, and a stripe of goat antihuman IgG in the “C” (control) location. The specimen of blood or plasma is added directly to the developer vial. For oral fluid testing, the oral fluid sample is collected by swabbing the gums with the paddle-shaped device. The test device is then added to the developer vial. If HIV antibodies are present in the specimen, they bind to the peptides causing a red line to appear in the test location. As the solution migrates further, it encounters the antihuman IgG control, and if an adequate specimen was added, a red line appears in the control location.
The test result should be read no sooner than 20 minutes and no later than 40 minutes after the test device is inserted into the developer vial. A red line at both the test and control location indicates a valid reactive test result; a red line only in the control location indicates a valid negative test result. The test is invalid and should be repeated with a new device if no line appears at the control location or if lines appear outside the areas indicated by the triangles [10].
Designed as a point-of-care HIV test, OraQuick® has been used in numerous settings including labor and delivery [11•], ambulatory clinical sites [12], emergency departments [13,14], hospital inpatient services [15] (Greenwald JL, unpublished data), correctional facilities [16], and for occupational exposures [17–19]. Additionally, OraQuick® has also been used by the military in battlefield operations [20].
Reveal™ G2 Rapid HIV-1 Antibody Test
On April 17, 2003, the FDA approved the Reveal™ Rapid HIV-1 Antibody Test to detect HIV antibodies in serum or plasma. In June 2004, it was superseded by the second generation Reveal™ G2 test, which incorporates an internal control [21]. Reveal™ G2 consists of a test cartridge and a proprietary colorimetric detection agent. Positive and negative external controls, which must also be reconstituted, are supplied with the kit. Reveal™ is considered reactive if both the red control line and central red test dot appear, negative if only the control line appears, and invalid if the control line does not appear (Fig. 2). The Reveal™ G2 only takes 3 minutes to run [22]. However because it requires serum or plasma from centrifuged blood samples and several reagent steps, it is classified as a moderate complexity test under CLIA and is usually performed in a clinical laboratory.
Uni-Gold Recombigen® HIV Test
The Uni-Gold Recombigen® HIV Test received FDA approval in December 2003 for testing whole blood, serum, and plasma for antibodies to HIV-1. It was waived under CLIA in 2004 for use with venipuncture and fingerstick whole blood specimens [23]. The device consists of a rectangular plastic test cartridge and a dropper bottle of buffer solution (Fig. 3). Peptides from the immunodominant region of the HIV-1 envelope are immobilized on a nitrocellulose strip in the test region. Reagents are also bound at the control region to indicate whether the test is functioning correctly, but these do not detect IgG and thus appearance of the control line does not validate that adequate patient specimen has been added. One drop of specimen is added to the specimen well on the test cartridge followed by four drops of wash buffer. The specimen combines with the colorimetric reagent and migrates along the nitrocellulose strip past the test and control regions. The test is read 10 to 12 minutes after specimen is added. A line in both the test and control regions indicates a reactive test; a line in only the control region indicates a negative test. When used with whole blood, the test is valid only if the control line is present and the sample well is red, indicating that an adequate blood sample has been added [24].
Multispot HIV-1/HIV-2 Rapid Test
The Multispot HIV-1/HIV-2 Rapid Test received FDA approval in November, 2004 [25]. Multispot is classified as a moderate complexity under CLIA, approved for use on fresh or frozen serum and plasma to both detect and distinguish HIV-1 from HIV-2. Multispot consists of a test cartridge and five reagents: specimen diluent, wash solution, conjugate, development reagent, and stop solution. The cartridge contains a membrane on which microparticles have been immobilized in four spots. Two of the spots consist of ecombinant and synthetic gp41 peptides to detect HIV-1 antibodies; one consists of synthetic gp36 peptides to detect antibodies to HIV-2; and the fourth spot consists of goat antihuman IgG as the internal control.
The test is considered positive for HIV-1 if the control spot and either or both of the HIV-1 spots turn purple, and positive for HIV-2 if the control and HIV-2 spots appear (Fig. 4). If purple appears in the control spot, the HIV-2 spot, and one or both of the HIV-1 spots, the test is considered HIV reactive (undifferentiated). In this case, the specimen may be tested by additional methods, which allow differentiation between HIV-1 and HIV-2. The test is negative when only the control spot appears. The absence of the control spot indicates an invalid result, regardless of any other spot pattern.
Rapid HIV Antibody Test Performance and Interpretation of Test Results
Like conventional EIAs, rapid HIV tests are screening tests. If performed correctly, they detect HIV antibodies with sensitivities similar to currently available EIAs [10,22,24–29] (Table 1). A negative rapid HIV test result requires no further confirmatory testing. False negative results, though rare, may occur in a person who has been acutely infected but who has not yet developed HIV antibodies. Therefore, any patient testing negative who
has had known or suspected exposure to HIV within 3 months should be instructed to retest 3 months after the exposure date [30]. Additionally, false-negative rapid HIV
test results have been observed in some patients receiving highly active antiretroviral therapy with undetectable virus in whom levels of HIV antibody have waned below the level of detection by the rapid HIV test [31].
A reactive result from any of the four rapid HIV tests is interpreted as a “preliminary positive” and requires confirmation by a more specific assay, typically a Western Blot (WB) or immunofluorescent assay (IFA) [10,22,24,25]. Performing a standard EIA screening prior to confirmatory testing is not required. However, if an EIA is performed, the specimen must still proceed to WB or IFA testing regardless of the EIA result. A positive WB or IFA confirms the diagnosis of HIV infection. If the con-firmatory test yields negative or indeterminate results, follow-up HIV testing should be performed on a blood specimen collected 4 weeks after the initial reactive rapid HIV test result [32•] as some patients newly infected with HIV may not have developed antibody levels sufficient to produce a positive WB or IFA [33].
Table 1 presents the test performances of US FDAapproved rapid HIV tests. It is important to note that because the test specificities are less than 100%, false positive rapid test results are an expected but rare event.
When testing low seroprevalence areas, a higher proportion of reactive tests will be false positives because there are few true positives in low-prevalence populations. The causes of falsely positive rapid HIV tests (ie, a reactive rapid HIV test with a negative or indeterminate confirmatory test) are poorly understood. Certain medical conditions may be associated with a slightly increased risk for false-positive OraQuick® rapid HIV tests (eg, hepatitis
A and B viruses, Epstein-Barr virus, multiparity, and the serologic presence of rheumatoid factor) [10].
Quality Assurance for CLIA-waived Rapid HIV Antibody Tests
Although CLIA-waived rapid HIV test devices are easy to use and can provide reliable results when the manufacturer’s directions are followed, mistakes can occur at any point in the testing process, including storage and testing area temperature, test kit shelf-life, specimen collection, test performance and results interpretation, referring specimens for confirmatory testing, managing confirmatory test results, etc. To reduce mistakes and to ensure that the FDA restrictions for sale of the test are followed, a site that performs rapid HIV tests must have a QA program in place before offering these tests.
In January 2003, the CDC convened a panel of experts including laboratory scientists and individuals from the FDA and the Centers for Medicare and Medicaid
Services to develop guidelines that outline the basic parts of a rapid HIV test QA program [32•]. The Quality Figure 3. Uni-Gold Recombigen® HIV test. A Rapid Review of Rapid HIV Antibody Tests Greenwald et al. 129 Assurance Guidelines for Testing Using the OraQuick® Rapid
HIV-1 Antibody Test are intended to assist a range of providers in developing policies, processes and procedures to ensure high quality HIV testing services. These guidelines include 1) the basics of a QA program for testing using OraQuick®, 2) an overview of government rules that apply to using this test, and 3) examples of forms/checklists that can be used to keep track of QA outcomes.
Counseling with Rapid HIV Antibody Tests
Counseling for patients choosing rapid HIV testing involves some differences compared with conventional testing, including assessing preparedness for clients to receive test results in the same session and explaining the meaning of preliminary positive results. Information can be provided either face-to-face or in a pamphlet, brochure, or video [34].
Patients with reactive rapid test results must be counseled in simple terms about the meaning of a reactive test. The provider must emphasize the need for a confirmatory test and schedule a return visit for results. Providers offering rapid HIV testing should be able to collect blood or oral fluid specimens on-site for confirmatory testing. All patients with reactive tests should be counseled on risk-reduction behaviors while awaiting the results of confirmatory testing. A simple message to convey this information could be “Your preliminary test result is positive, but we won’t know for sure if you are infected with HIV until we get the results from your confirmatory test. In the meantime, you should take precautions to avoid transmitting the virus” [34]. The New York State Department of Health AIDS Institute has also created guidelines for how to discuss reactive results stratifying the language based on the patient’s level of risk for HIV infection. For clients at high risk, the guidelines suggest saying “Based on your risk factors, it is highly likely that the preliminary test result is correct and that you have HIV” (emphasis added). For those at low risk, the phrase “quite likely” is recommended, and for those with no admitted risk factors, they advise informing them “There is a chance that this result could be a false positive” [35].
Physicians and counseling staff may be apprehensive about rapid testing specifically with regards to the ability to handle preliminary positive test results at any time. Data from RESPECT-2, a large, randomized, controlled trial that compared different forms of HIV testing and risk-reduction counseling in clients at sexually transmitted disease (STD) clinics in the United States, found that after gaining experience in the field, the majority of counselors preferred rapid testing, felt that rapid HIV testing sessions resulted in enhanced counseling, and felt that it was more convenient for both clients and counseling staff [36]. Although some have expressed concern about how counselors and clients will deal with discussing and understanding reactive results [37], others have noted that providers have extensive experience managing preliminary positive test results (eg, abnormal mammograms that require biopsies and abnormal pap smears that require colposcopy) [38] and studies of rapid testing have demonstrated good client understanding of results [39].
Providing HIV counseling and testing may be challenging in some health care settings. Because the average primary care office visit in the United States is less than 18 minutes long [40], even the “brief” counseling protocol of RESPECT-2 could take up an entire office visit. In these situations, alternative procedures for HIV counseling with rapid testing should be considered, eg, providing information either in a face-to-face meeting with a counselor or in a pamphlet, brochure, or video [34].
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