Tools
HLA-B*57:01 Testing
Introduction
HIV targets the body’s immune system—specifically CD4 T cells. The virus is transmitted through bodily fluids but can remain undetected in its host for years, causing a diverse array of complications. During the progression of HIV infection, a patient’s CD4 lymphocyte count will decrease. Individuals with HIV who do not receive treatment can develop AIDS once their CD4 lymphocyte count decreases; these patients are highly susceptible to opportunistic infection. Patients that develop AIDS and remain untreated have a 1 to 2 years prognosis of survival.[1]
Due to the loci of infection, managing the virus uses a multifaceted approach. The core of HIV treatment is highly active antiretroviral therapy (HAART). In most patients, HAART includes 2 nucleoside reverse transcriptase inhibitors (NRTIs) and an integrase strand transfer inhibitor (InSTI).[2] These regimens seek to interrupt viral replication and slow disease progression. Abacavir is an NRTI.[3]
Abacavir causes a hypersensitive response in 5% to 8% of patients treated. Cytotoxic T cells mediate the hypersensitivity reaction to abacavir. Hypersensitivity reactions (HSR) can occur within the first 6 weeks of treatment if genetic prescreening is not conducted. If the drug is continued despite worsening symptoms, the severity of the HSR can escalate over time. Symptoms of an HSR typically include at least 2 of the following: fever, rash, fatigue, cough, difficulty breathing, and gastrointestinal issues such as nausea, vomiting, or abdominal pain.[4][5]
If an HSR is suspected, abacavir should be discontinued immediately. Should the symptoms of a clinically diagnosed HSR improve after stopping abacavir, it is crucial to avoid reintroducing the drug, as this can lead to immediate and potentially life-threatening reactions, including anaphylaxis and death.[6] The signs and symptoms of abacavir hypersensitivity are nonspecific and may be confused with other conditions frequently encountered in HIV-positive patients, such as infections, immune restoration disease, and hypersensitivity reactions to other medications.[7] Therefore, understanding the mechanism and predictive power of the major histocompatibility complex, class I, B 57:01 (HLA-B*57:01) allele concerning hypersensitive reactions is critical in shaping the best clinical practices.[8]
Specimen Collection
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Specimen Collection
Specimen collection uses whole blood and buccal mucosa swabs. The volume of the sample/specimen should be 7 mL in a whole blood–lavender-top (ethylenediaminetetraacetic acid; EDTA) tube and 4 buccal swabs in a sealed buccal swab envelope kit. The EDTA prevents clotting and preserves the cellular components essential for accurate testing.[9] After collection, the tube should be gently inverted several times to ensure proper mixing of the blood with the EDTA. The specimen must be stored at room temperature and processed as soon as possible to prevent degradation of the cells.[10]
Care must be taken during the blood collection to prevent hemolysis, which can occur if the blood is drawn too forcefully or if the collection tube is shaken vigorously. Hemolysis can release intracellular components that may interfere with the test results.[11] When collecting buccal swabs, avoiding touching the swab tip with fingers or any nonsterile surfaces is essential to reduce the risk of contamination, which can compromise the sample's integrity.[12]
Clinicians should ensure buccal swabs are completely dry before sealing them in an envelope. Failing to do so can result in the degradation of cellular material, potentially leading to inaccurate test results. Patients should not eat, drink, smoke, or chew gum for 30 minutes before specimen collection.[13]
Procedures
After establishing a link between the HLA-B*57:01 allele and abacavir, monitoring for the allele and hypersensitive response became routine in antiretroviral therapy, accomplished through genetic screening. Testing for HLA-B*57:01 most commonly uses a blood or saliva sample for sequence-based genotyping using a polymerase chain reaction.[8] Following these tests, the clinician will know if the patient is positive for the HLA-B*57:01 allele and be able to alter the treatment accordingly.
For patients with the HLA-B*57:01 allele, safely and conclusively diagnosing hypersensitivity is another necessary step in determining drug efficacy. To confirm hypersensitivity, an epicutaneous patch test is administered after the patient has had exposure to abacavir. A skin patch must occur after the immune system has been exposed to the drug's effects because a rechallenge will produce a measurable response. Skin patch testing uses concentrations of 1% and 10% abacavir in petrolatum along with a control without abacavir. These patches can then be read 24 and 48 hours after application. A positive hypersensitive response is a palpable cutaneous response on the skin surface where abacavir was introduced.[14][15][16]
Implementing comprehensive quality assurance processes for HLA-B*57:01 testing is essential for accurately identifying patients at risk for abacavir hypersensitivity.[17] By focusing on routine equipment calibration, continuous staff training, participation in proficiency testing programs, adherence to standard operating procedures, and diligent documentation, laboratories can enhance the reliability of their testing results. These quality control measures improve patient safety and contribute to the overall quality of care in pharmacogenetic testing.[18][19]
Participation in regular external quality assessment surveys allows labs to monitor their performance over time. External quality assessment programs distribute blinded samples to participating laboratories for HLA-B*57:01 typing. This allows for an objective assessment of each lab's performance in accurately detecting the presence or absence of the allele. Any deviations from expected results can prompt investigations into potential sources of error and implementation of corrective actions.[20][21]
Indications
Patients with HIV should be screened for HLA-B*57:01 when preparing an antiretroviral therapy drug regimen, especially when considering abacavir. Even if previously tolerated, screening should happen before restarting abacavir therapy if the HLA-B*57:01 status is unknown. Implementing HLA-B*57:01 screening in clinical practice has been shown to reduce the incidence of abacavir hypersensitivity reactions from 1.3% to 0.2% over several years, demonstrating the clinical benefit of genetic screening before treatment initiation.[17][8]
In addition to its link with abacavir hypersensitivity reactions, HLA-B*57:01 has also been associated with liver injury caused by flucloxacillin. While individuals with the HLA-B*57:01 allele have a greater than 40-fold increased risk of liver injury compared to those who without the allele, flucloxacillin-induced hepatotoxicity remains very rare, occurring in fewer than 1 in 5000 cases. This incidence is significantly lower than that of abacavir hypersensitivity reactions. Therefore, routine screening for HLA-B*57:01 to assess the risk of flucloxacillin-induced liver injury is not commonly performed.[22][23]
Normal and Critical Findings
Screening for the presence of the HLA-B*57:01 allele should always be conducted to avoid possible morbidity from allergic reactions. The genotype results for the HLA-B*57:01 allele can be classified as either positive or negative, with no intermediate phenotypes due to the codominant expression of HLA genes. A positive result indicates that the patient has at least 1 copy allele; patients may be heterozygous or homozygous.[24]
In patients with a positive result, abacavir is contraindicated. Due to the implications of positive results for the HLA-B*57:01 allele, using abacavir in treatment is avoided. Instead, clinicians should substitute a different NRTI in the therapeutic regimen. Conversely, a negative result signifies that the patient does not possess the HLA-B*57:01 allele. If the patient is negative for the allele, clinicians can safely resume abacavir as a component of the patient's regimen.[8]
However, clinicians should note that a negative result does not eliminate the risk of developing hypersensitivity reactions to abacavir. Therefore, all patients should be monitored closely, following standard medical practices. In rare cases where HLA-B*57:01 testing is not available due to geographically low prevalence of the allele, clinicians may prescribe abacavir under close observation for the first 6 weeks, as the median onset time of HSR is typically 1 to 2 weeks.[25]
Interfering Factors
In studies of abacavir hypersensitivity reaction or AHR, researchers found that a significant portion of hypersensitive diagnoses were false positives using epicutaneous patch testing. HLA-B*57:01 screening has shown to have a 100% negative predictive value.[26] Interestingly, although all patients who develop hypersensitivity have the HLA-B*57:01 allele, not all individuals with that genotype develop hypersensitivity. Mouse models have supported the hypothesis that CD4 T cells play a role in building tolerance for the altered antigen-presenting complex through cytotoxic lymphocyte-driven hypersensitive response.[27][28]
Clinicians must use patch testing to confirm the diagnosis of hypersensitivity in patients. In blind comparative trial regimens, 2% to 7% of patients were diagnosed with hypersensitivity without receiving abacavir.[17] This discrepancy in true positive hypersensitivity recognition is due to the symptoms of hypersensitivity being nonspecific and difficult to distinguish from other ailments that could accompany the introduction of a new drug regimen.
Complications
Hypersensitivity can manifest as a variety of symptoms. The most common AHR symptoms include fever, chills, rash, gastrointestinal, and respiratory symptoms. Patch testing is often used to confirm hypersensitivity and will cause a palpable cutaneous response on the skin.[29]
Clinical Significance
As a NRTI, abacavir competes with deoxynucleotides in host cells to prevent the creation of double-stranded viral DNA, thereby limiting HIV replication.[30] Hypersensitivity can arise when individuals with antigen-presenting complexes linked to HLA-B*57:01 perform direct noncovalent bonding with abacavir. Once bonded, the surface complex is altered and is then recognized as foreign by cytotoxic lymphocytes. This interaction results in the release of inflammatory cytokines that facilitate the hypersensitive response.[14] Although it is primarily the parent drug that triggers hypersensitivity, it is hypothesized that abacavir metabolites may contribute to the immune response by generating altered peptides. These peptides might be presented on HLA molecules, potentially amplifying the immune response in some cases, but this is not considered the main pathway.[25][31]
Due to the severity of AHR, clinicians should conduct screenings for the HLA-B*57:01 allele to avoid a hypersensitive response in patients predisposed to this outcome. Clinicians must consider the patient’s HLA-B*57:01 status when selecting a regimen. The recommendation is that patients receive an alternative NRTI to abacavir if they have the HLA-B*57:01 allele to avoid the possibility of a hypersensitive response.
Since rechallenging the immune system after a hypersensitive response can produce extreme and life-threatening symptoms, the use of abacavir in patients who may have had an allergic reaction previously should be cautiously considered. Skin patch testing can also be a consideration in these cases since it is used exclusively in patients who have already taken abacavir and serves as a localized challenge instead of a systematic reintroduction. If clinicians observe these protocols and recommendations, clinicians and patients can make an informed selection of the components of their antiretroviral therapy. For most, abacavir is very effective in concert with an additional NRTI and an InSTI, but for a small percentage of genetically predisposed individuals, abacavir can result in a harmful hypersensitive reaction.
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