HIV-1 Viral Load After Leukodepletion (2008)

To the Editor: More than 40 million people are infected with HIV, the causative agent of AIDS, and more than 95% of them are in developing countries. The screening of donated blood and education about the danger of unprotected sex were the main factors to herald the explosion of the HIV pandemic. The serology-based screening of donated blood is not 100% risk-free, however. This is because antibodies against HIV are not seen in serum immediately after exposure; it may take up to 8 weeks, the “window period,” before they are seen.1 In this window period, the viremia is high and then drops as the cytotoxic CD8 T lymphocytes develop and an individual viral load set point is reached during chronic infection. Viral set points differ greatly among individuals and are used to predict disease progression.2 The danger of HIV being transmitted through blood transfusion during the window period is practical. In the United Kingdom, the statistical projections of potential window period-infected donations suggest an occurrence of approximately 1 in 2.5 million.3 It is estimated to be higher in South Africa, where it ranges from 1.1 to 3.9 per 100,000 units, with a likely estimate of 2.2 per 100,000 units.4 The first advance in this regard was the inclusion of P24 viral antigen screening, which has been shown to reduce the diagnostic window of HIV infection by 6 days compared with the use of a “third-generation” anti-HIV enzyme immunoassay alone.5 The second advance was the screening for viral RNA by reverse transcriptase polymerase chain reaction (RT-PCR) in pooled blood samples. The risk of HIV transmission can be further reduced 45% to 72% by nucleic acid amplification technology (NAT) screening.6 In the United States, NAT has reduced this risk of HIV transmission through blood donation to approximately 1 in 1,900,000.7 The technology of RT-PCR is expensive, however, and cannot be used in developing countries, where the largest bulk of HIV incidence is concentrated. For such countries, some researchers have advocated the use of white blood cell (WBC) filters to reduce the likelihood of transmission of infective agents, such as cytomegalovirus (CMV), HIV, and Leishmania.8 The principle of the WBC filtration (leukodepletion) is based on the assumption that HIV and other intracellular infective agents reside in CD4 T lymphocytes and macrophages. Blocking these reservoirs of infective agents from transmission to blood recipients would decrease the load of transfused agents, and this might reduce the infectivity of blood and blood products, especially in window period when conventional serologic tests are not reliable. Cervia et al9 have done a comprehensive review on leukodepletion and its role in attenuation of infection risks among transfusion recipients. We have undertaken a study to investigate the amount of HIV-1 viral load reduction after leukocyte filtration. We have initiated this pilot study to evaluate HIV-1-infected WBC filtration using blood samples from patients at Sultan Qaboos University Hospital (SQUH). We have performed the investigation using extracted blood from known HIV-positive patients who are not receiving highly active antiretroviral therapy (HAART). With ethical approval and patient consent, blood donated from each patient was collected using the blood collection pack (PackPure WB, Baxter, Berkshire, UK) system. The system is composed of 2 450-mL packs connected with a flexible screen filter with a polyvinylidene difluoride membrane (1.0 μm and 0.65 μm). The movement through the filter is gravity dependent. A 5-mL blood sample was taken before filtration from the blood collection pack into an ethylenediaminetetraacetic acid (EDTA) tube for viral load measurement. The blood moved with gravity from the collection pack to another pack set at a lower level through a filter. The pack containing the filtered blood was disconnected from the filter and collection pack. A 5-mL blood sample was removed after filtration from the filtered pack into the EDTA tube for viral load measurement. Viral load measurements in plasma of the unfiltered and filtered blood samples were run at the same time using the COBAS AMPLICOR (Roche Molecular Diagnostics, Pleasanton, CA). The HIV-1 RT-PCR assay was based on the concentration of RNA at high-speed centrifugation, alcoholic extraction of RNA from the plasma samples followed by reverse transcription using Thermus thermophilus, and the eventual PCR assay as per the manufacturer's instructions. The amplicons were detected using an enzyme-linked immunosorbent assay (ELISA) and quantified as per a standard curve. The tests were repeated 3 times for every sample. Two patients met the criteria of having HIV and still not being on HAART. The samples were tested first serologically for HIV-1 by ELISA and then confirmed by Western blot analysis. We noted a nonsignificant increase of virus load rather than the expected decrease of virus load after leukodepletion (Table 1). The original viral loads for both patients were high 78,000 (±400) copies/mL and 69,500 (±200) copies/mL, respectively. There was no decrease in the viral load of both patients after filtration; the viral loads of filtered blood were 79,950 (±350) copies/mL and 70,000 (±200) copies/mL, respectively.TABLE 1: Viral Load Before and After WBC Filtration for Samples A and BIn the ultrasensitive PCR assay employed, the variability of the standards used ranges from 63 to 1000 for the lower limit of the assay range and from 5100 to 82,000 for the upper limit of the assay range. This means that the significance of the results is limited to the log phase only. There is still the possibility that the slight increase in the postsamples is attributable to the pressures of filtration, which could cause leukocyte lyses releasing more viruses. The leukodepletion procedure is labor-intensive and requires extra time compared with conventional techniques, which may add to delaying the freezing time of donated blood. Leukodepletion is usually achieved by filtration of whole blood or blood components or by certain apheresis techniques without filtration, and most filters provide 3- to 4-log removal of leukocytes.10,11 HIV transmission through blood and blood product transfusion is still a real problem, especially in countries with a high rate of infection and with limited resources.9 A search for alternative methods for the reduction and/or elimination of viral load before blood transfusion is highly needed. Although we report here that leukodepletion does not reduce HIV-1 viral load, we would recommend that other studies with a larger sample size and variable viral load be conducted. ACKNOWLEDGMENT The authors acknowledge the help of Frank Emmanuel Serebour during PCR testing. Zakariya Al-Muharrmi, MD* Ali A. Al-Jabri* Ghada Al-Rawahi* Euan M. Scrimgeor† A. Al-Marhobi‡ *Department of Microbiology and Immunology College of Medicine and Health Sciences Sultan Qaboos University Muscat, Oman †Department of Medicine College of Medicine and Health Sciences Sultan Qaboos University Muscat, Oman ‡Blood Bank College of Medicine and Health Sciences Sultan Qaboos University Muscat, Oman