Diagnostic Testing for HIV Type 1 RNA in Seronegative Blood

Transcription

1 Coagulation and Transfusion Medicine / DIAGNOSIS OF EARLY HIV INFECTION Diagnostic Testing for HIV Type 1 RNA in Seronegative Blood Detlef Ritter, MD, 1,2 James Taylor, 1 Richard Walkenbach, 3 Michael Creer, MD, 1 and Max Q. Arens, PhD 4 Key Words: HIV; polymerase chain reaction; PCR; Diagnostic Abstract We studied the feasibility of routine diagnostic testing for HIV-1 RNA at a publicly funded testing site. HIV-1 RNA was determined with a commercial polymerase chain reaction assay in pooled seronegative blood samples submitted for HIV testing to a public health laboratory. Recovery of HIV-1 RNA from the samples was estimated as at least 8% of viral RNA that was found in freshly prepared plasma. We estimated that screening for HIV-1 RNA in serum pools would result in the identification of blood specimens from more than 95% of acutely infected patients. The frequency of HIV-1 RNA in seronegative blood samples was estimated to be between 19 and 601 per 10 6 submitted specimens. The ratio of HIV-1 RNA positive and seronegative samples to specimens with HIV-1 antibodies confirmed by Western blot was estimated to be between 0.2% and 6.6%. The reagent costs for identifying 1 HIV-infected blood sample were 10-fold higher with the commercially available HIV-1 RNA assay compared with the HIV antibody enzyme-linked immunosorbent assay. Diagnostic testing for HIV-1 RNA may be warranted in high-risk populations since acutely infected patients may benefit most from antiretroviral therapy and are thought to contribute disproportionately to the HIV epidemic. Identification of patients with HIV infection is based on the detection of circulating antibodies to various viral proteins. Patients serum samples that are reactive by enzyme immunoassays must be confirmed by Western blot. During the early stage of HIV infection, detectable antibody may not have formed, and antibody-based assays may be unreliable for the detection of HIV infection. 1 Patients with acute HIV infection have high circulating levels of viral particles. In these patients, HIV infection may be detected by assays that are based on the detection of viral antigen or viral RNA. Assays for p24 antigen are used for the detection of early HIV infection in blood donors. 2 Furthermore, blood services in the United States have decided to embark on nucleic acid testing for blood donors. The most sensitive assays for HIV virus are based on the detection of viral RNA. 1 In the present study, we used a commercially available polymerase chain reaction (PCR) assay to estimate the frequency of HIV-1 RNA in seronegative blood samples that had been submitted to the Missouri Department of Health State Public Health Laboratory (Missouri Public Health Laboratory) for HIV testing. Methods Collection, Transport, and Processing of Blood Specimens at the Missouri Public Health Laboratory Blood samples were collected in blood collection tubes (Vacutainer, Becton Dickinson, Franklin Lakes, NJ) without additives and submitted to the Missouri Public Health Laboratory at ambient temperatures via regular mail or by private mail couriers. More than 90% of the blood samples that are received by the state laboratory for HIV testing consisted of clotted whole blood specimens. The remaining 128 Am J Clin Pathol 2000;113: American Society of Clinical Pathologists

2 Coagulation and Transfusion Medicine / SPECIAL ARTICLE specimens were serum samples. On arrival of the samples at the laboratory, cellular components were removed from whole blood specimens by centrifugation, and serum samples were tested during the same day or stored at 4 C for testing at the next day. All serum samples were screened with an enzyme-linked immunosorbent assay (ELISA) for antibodies to HIV-1 (Vironostika HIV-1 Microelisa System, Organon Teknika, Durham, NC). Serum samples that were positive repeatedly by ELISA were tested by Western blot (Novapath HIV-1 Immunoblot, Bio-Rad Laboratories, Hercules, CA). Seronegative serum specimens were stored at 20 C for a period of 1 to 2 months. We obtained approval from the institutional review board to study blood specimens for which HIV antibody testing had been completed. Serum samples from consecutive seronegative blood samples that had been submitted to the Missouri, Public Health Laboratory during the months July and November 1997 were selected for the study and stored at 75 C for up to 1 year. Analysis of Blood Specimens Submitted to the Missouri Public Health Laboratory HIV-1 RNA was determined in serum pools. Small pools were generated by combining aliquots of 0.1-mL volume from 10 original samples. Aliquots containing 0.1 ml from 10 small pools were used to make large pools from 100 original donors. HIV-1 RNA was measured in large serum pools with the Amplicor HIV-1 Monitor test (Roche Diagnostic Systems, Branchburg, NJ) using a protocol that decreased the detection limit from 400 copies per milliliter to 40 copies per milliliter. 3 Initially, viral particles were pelleted by high-speed centrifugation of specimens for 1 hour at 25,500g. The RNA was extracted with modified reagent volumes as described previously. 3 Reverse transcription, amplification by PCR, and detection were performed according to the manufacturer s instructions. 4 The linear range of the ultrasensitive assay was 40 to 50,000 copies per milliliter. To resolve which sample(s) caused a positive result in large pools, HIV-1 RNA was measured in each of the 10 small pools. HIV-1 RNA was determined in original samples from small pools that tested positive with the PCR assay. HIV-1 RNA positive blood samples were retested with HIV- 1 antibody ELISA and Western blot as described in the previous section. All statistical analysis was performed with the InStat Program (GraphPad Software, San Diego, CA). Recovery of HIV From Spiked Whole Blood or Serum Specimens One hundred milliliters of fresh citrated whole blood was obtained from 1 HIV seronegative donor. HIV-1 virus was derived from plasma of patients with HIV-1 RNA titers between 2 and 3 million copies per milliliter. The fresh citrated blood was split into 2 portions of equal volume. Each of the 2 portions containing 50 ml of citrated whole blood was spiked with 2 ml or 0.2 ml of HIV-1 containing plasma, respectively. The spiked whole blood was split into 1-mL aliquots. Aliquots that served as plasma controls were centrifuged after incubation at room temperature for 1 hour, and separated plasma was stored at 75 C for HIV-1 RNA testing. The remaining spiked blood was clotted by the addition of 33 µl calcium chloride (at a 250-mmol/L concentration) to each of the aliquots. Two clotted aliquots were centrifuged, and serum was frozen 1 hour after addition of calcium chloride. For each of the 2 pools that contained different viral concentrations, duplicate aliquots were kept at 4 C or 25 C for 1, 3, or 5 days. Duplicate aliquots also were kept at higher temperatures with an initial incubation at 45 C for a period of 8 hours followed by storage at 37 C for the remaining time. Upon completion of sample incubation, serum samples were separated from cellular components by centrifugation and kept at 75 C until further analysis. For recovery studies of HIV-1 in serum samples, each aliquot containing HIV-spiked citrated whole blood was cetrifuged 1 hour after addition of calcium chloride, and serum was separated from cellular components. The serum samples were subjected to the same temperature conditions as described for clotted whole blood. On completion of incubation, serum samples were kept at 75 C until analysis. Data are given as mean ± SEM unless otherwise stated. Results Serologic Status of HIV-1 in Blood Samples The Missouri Public Health Laboratory performs screening and confirmatory testing for HIV for a variety of institutions and private practices. During the study period, blood samples were submitted from the locations listed in Table 1. The highest frequency of confirmed HIV seropositivity was found in samples that were submitted by governmental institutions. Stability of HIV-1 Virus in Clotted Whole Blood and in Serum The preferred specimen for determination of HIV-1 RNA is freshly isolated plasma. 4 However, most of the samples that were submitted to the Missouri Public Health Laboratory consisted of clotted whole blood specimens that were mailed at ambient temperatures. We determined the recovery of HIV-1 in clotted whole blood and in serum samples that had been spiked with various concentrations of HIV particles and kept at temperatures between 4 C and 45 C for a period of up to 5 days. Plasma that was prepared from HIV-spiked anticoagulated whole blood had 116,846 American Society of Clinical Pathologists Am J Clin Pathol 2000;113:

3 Ritter et al / DIAGNOSIS OF EARLY HIV INFECTION Table 1 Origin and Frequency of Seropositive Blood Samples Submitted to the Missouri Department of Health State Public Health Laboratory During the Testing Period Specimen Origin No. of Specimens No. (%) Western Blot Reactive Private hospitals, laboratories, and physician offices 1, (0.93) STD prevention programs 5, (0.85) Drug treatment programs (0.72) Local public health departments 2,050 6 (0.29) Family planning clinics (0.00) Governmental institutions * 1, (1.96) Total 12, (0.90) STD, sexually transmitted disease. * Department of Corrections, Department of Mental Health, Veterans Affairs hospitals and university student health centers. ± 3,548 copies of RNA per milliliter. In contrast, recovery of viral RNA from freshly isolated serum samples was significantly lower at 32,834 ± 997 copies per milliliter (unpaired t test, P <.003) as previously described. 5 Incubation of clotted whole blood at room temperature or at 37 C to 45 C for up to 5 days did not result in significant changes of HIV-1 RNA concentrations (analysis of variance, nonsignificant) compared with immediately prepared (day 0) and frozen serum Figure 1. In contrast, HIV-1 RNA concentrations decreased significantly (analysis of variance, P <.001) in refrigerated clotted whole blood samples. On days 3 and 5, 9,474 ± 297 and 9,777 ± 325 copies per milliliter of viral RNA were recovered from spiked and refrigerated clotted whole blood samples, respectively. This corresponds to a recovery of 8% compared with the amount of HIV-1 RNA in freshly prepared plasma samples. In contrast with clotted whole blood, the incubation of HIV-containing serum at room temperature or at 4 C did not significantly affect the recovery of HIV-1 RNA particles Figure 2. Storage of HIV-containing serum at 37 C to 45 C resulted in a significant decrease of recovered viral particles from 33,058 ± 9,181 on day 0 to 3,212 ± 476 copies per milliliter on day 5 (Kruskal-Wallis test, P <.01). Our test configuration allowed us to identify original samples with viral RNA concentrations greater than 4,000 copies per milliliter. When anticoagulated whole blood specimens that had been spiked with HIV resulting in HIV-1 RNA plasma concentrations of approximately 100,000 copies per milliliter were clotted and incubated under various conditions, more than 4,000 copies per milliliter were recovered in all isolated serum samples. These results suggest that our assay configuration would have detected clotted whole blood samples with HIV-1 RNA concentrations greater than 100,000 copies per milliliter under all of the tested storage conditions. In contrast, blood samples that were spiked with lower concentrations of HIV-1 RNA (plasma concentration: 11,876 copies per milliliter) would be identified with our test Fraction of HIV Recovered Day configuration only if the blood samples were incubated between 37 C and 45 C. Frequency of HIV-1 RNA in HIV Seronegative Blood Samples Serum samples were pooled to decrease the number of samples to be tested. We screened 120 pools, representing 12,000 seronegative serum samples, for the presence of HIV- 1 RNA. Two large pools were identified to contain HIV-1 RNA Table 2. Testing of small pools and original serum 4 C 22 C 37 C Figure 1 Recovery of HIV-1 RNA from clotted whole blood specimens compared with freshly prepared frozen plasma. Two citrated whole blood samples from a seronegative donor were spiked with varying amounts of HIV-1, resulting in final plasma HIV-1 RNA concentrations of 116,846 ± 3,548 (triangles) and 11,876 ± 1,799 (squares) copies per milliliter (average ± SEM, N = 2), respectively. The spiked whole blood was clotted with calcium and stored under conditions as shown in the figure. After storage, cellular components were removed, and HIV-1 RNA concentrations were determined in duplicate serum samples with a commercial HIV-1 RNA polymerase chain reaction procedure. HIV-1 RNA results were tested for significant differences by analysis of variance. Asterisk indicates P <.001. * 130 Am J Clin Pathol 2000;113: American Society of Clinical Pathologists

4 Coagulation and Transfusion Medicine / SPECIAL ARTICLE Table 2 HIV-1 RNA Concentrations and Antibody Test Results in Original Specimens From 2 HIV-1 RNA Containing Pools Pool 1 Pool 2 Concentrations of HIV-1 RNA in positive pools, copies per milliliter 4, No. of HIV-1 RNA positive serum samples per pool 1 1 Concentrations of HIV-1 RNA in original samples, copies per milliliter 339,548 39,138 HIV-1 ELISA and Western blot in HIV-1 RNA positive samples Negative Negative ELISA, enzyme-linked immunosorbent assay. Fraction of HIV Recovered Day samples resulted in the identification of 1 HIV-1 RNA positive serum sample from each of the 2 pools that had been positive for HIV-1 RNA. The concentration of HIV-1 RNA increased proportionately in small pools (41,421 and 2,903 copies per milliliter) and in the original samples (Table 2). We retested the 2 HIV-1 RNA positive serum samples for antibodies to HIV viral proteins. Both samples were confirmed to be nonreactive with the HIV-1 ELISA and Western blot, thus excluding the possibility of the accidental inclusion of an HIV antibody positive sample into the pool of seronegative blood specimens that we studied. A total of 12,109 samples were submitted to the public health laboratory during the collection period. Of these, 109 samples were identified and confirmed to contain antibodies against HIV viral proteins. The proportion of blood samples that tested positive by HIV-1 RNA only was 1.8% of HIV-1 4 C 22 C 37 C Figure 2 Recovery of viral RNA from serum samples in comparison with freshly prepared frozen plasma. Anticoagulated whole blood was spiked with HIV-1 resulting in plasma HIV-1 RNA concentrations as described in Figure 1. Blood was clotted with calcium, and cellular components were separated by centrifugation. Aliquots of serum samples were incubated as indicated in the figure, and HIV-1 RNA concentrations were determined in duplicate serum samples after incubation. Significant differences determined by analysis of variance are marked with an asterisk (P <.05). * * antibody-positive samples Table 3. The detection rate of the HIV-1 RNA assay was 2 per 12,000 or 167 per 10 6 seronegative blood samples. Discussion Publicly funded testing sites fulfill an important function in the monitoring for infectious diseases such as HIV. The Missouri Public Health Laboratory tests samples from a substantial number of high-risk persons since the frequency of HIV antibody-positive blood samples is 2 to 3 times higher than the prevalence of HIV infection in the general US population. 6 It is well known that HIV ELISA assays and Western blot tests are insensitive for the detection of early HIV infection. 1,7-9 The window period between infection and seroconversion has been estimated to be 22 days (mean, 22 days; 95% confidence interval: 9-34 days) for third-generation antibody assays. 1 Other markers of HIV infection can further reduce this window period during which detection of viral markers is impossible. Assays for p24 antigen are thought to reduce the window period by 6 days, whereas HIV-1 RNA assays decrease the window period by 11 days. Blood banks have been required to include testing for p24 antigen into the mandated screening procedures of blood products for infectious diseases. 2 Testing of blood products for HIV-1 RNA will be implemented in the near future, resulting in a further decrease of the window period. In contrast with blood donations, routine diagnostic screening of blood specimens with p24 antigen assays is not performed by the US Public Health Service, since the reduction of the window period by 6 days is thought to result in the identification of only a few additional cases of HIV. 2 However, the benefit of virus detection assays most likely is much larger than previously estimated by the US Public Health Service. 2 First, state public health laboratories frequently use first-generation viral lysate assays for screening of HIV antibodies since these assays provide excellent correlations between ELISA and Western blot results. However, the average window period for viral lysate assays is approximately 42 days and considerably longer than described for third-generation assays. 1 Second, the technologic American Society of Clinical Pathologists Am J Clin Pathol 2000;113:

5 Ritter et al / DIAGNOSIS OF EARLY HIV INFECTION Table 3 Frequency of HIV-1 RNA and HIV-1 Antibody in 12,109 Tested Blood Samples Number 95% Confidence Interval Confirmed by Western blot Frequency of confirmed HIV-1 antibody 9,002 per ,350-10,818 Seronegative samples with HIV-1 RNA Frequency of HIV-1 RNA in seronegative blood samples 167 per Ratio of HIV-1 RNA containing seronegative samples to specimens 1.8% with confirmed HIV-1 antibody improvement of Western blot tests has not kept up with the development of third-generation HIV antibody assays. Patients with early HIV infection with positive results by third-generation ELISAs frequently have nonreactive Western blot results owing to the insensitivity of Western blot assays for IgM antibodies. 1,7-10 Assay results that have not been confirmed by Western blot often are considered to be falsepositive, and most of these patients will be lost owing to lack of follow-up testing. Third, assays for HIV-1 RNA have substantially lower limits of detection than p24 antigen assays, resulting in a further reduction of the window period. 1 We studied whether the implementation of an HIV-1 RNA PCR assay would be suitable for routine screening of blood specimens that were submitted to the Missouri Public Health Laboratory. We tried to address the following areas: technical difficulties to be solved, benefits for the population to be tested, and costs associated with test implementation. First, we studied whether blood specimens currently submitted to the Missouri Public Health Laboratory would be suitable for HIV-1 RNA testing. Commercially available PCR tests for HIV-1 RNA require plasma samples to be separated from cellular components within 6 hours of phlebotomy. 4 In contrast, most blood specimens that are submitted to state public health laboratories for HIV testing consist of clotted whole blood that has been exposed to different temperatures for various periods before testing. We found that the HIV-1 RNA level was decreased in clotted blood specimens that were exposed to conditions similar to those encountered by blood samples that are submitted to the Missouri Public Health Laboratory. The apparent differences of viral RNA contents in stored whole blood and serum samples at different temperatures may be due to many factors, including the possibility of continued viral replication in the remaining WBCs, the preferred adherence of viral particles or RNA to cellular elements at lower temperatures, and enzymatic disruption of viral RNA at higher temperatures. The recovery of viral particles in clotted whole blood was at least 8% of the HIV-1 RNA concentration that was measured in freshly prepared plasma. Next, we estimated the limit of detection that would be required for HIV-1 RNA assays to identify early HIV infection. Blood samples from patients with acute HIV infection contain high concentrations of viral particles. Of the specimens from acutely infected patients, 95% have viral concentrations between 10 5 and 10 8 copies per milliliter. 11,12 Therefore, assays with limits of detection greater than 10 3 copies per milliliter will identify 95% of samples that are submitted by patients with acute HIV infections. Since the limits of detection of the currently available HIV-1 RNA PCR assays are between 10 and 10 2 copies per milliliter, tests can be performed on pools of blood samples without compromising their ability to detect HIV in the of blood of acutely infected patients. However, newly infected patients who have not yet developed a viral syndrome may not have circulating viral concentrations that are high enough to be detected in pooled blood samples. To estimate possible benefits resulting from screening for HIV-1 RNA, we determined the frequency of HIV-1 RNA in seronegative blood samples that had been tested by the Missouri Public Health Laboratory. We identified 2 HIV-1 RNA positive samples during the screening of pools from 12,000 seronegative samples, suggesting that the true rate of HIV-1 RNA positive samples is between 19 and 601 per 10 6 seronegative specimens (95% confidence interval). The portion of HIV-infected blood samples that were not detected by the HIV-1 antibody test was estimated to be between 0.2% and 6.6%. The detection rate of the HIV-1 RNA assay may vary with the population to be tested. In a similar study performed in Switzerland, the screening of 10,692 seronegative blood samples with an HIV-1 RNA PCR assay resulted in the identification of 5 HIV-1 RNA positive patients who would not have been detected by conventional testing for HIV antibodies. 13 All of the 5 HIV-1 RNA positive patients seroconverted during followup testing. Furthermore, the incidence of HIV infection in the United States has been estimated to be low in cities of the Midwest compared with large eastern metropolitan areas, 6 suggesting that the results obtained in our study represent a rather conservative estimate of yields that may be expected in other regions of the United States. We also compared the detection rate of the HIV-1 RNA assay obtained in the present study with the detection rate 132 Am J Clin Pathol 2000;113: American Society of Clinical Pathologists

6 Coagulation and Transfusion Medicine / SPECIAL ARTICLE projected for blood donors. Testing of blood from low-risk blood donors with the HIV-1 RNA assay has been estimated to lead to the additional detection of 1 HIV-infected donation per 10 6 units of donated blood compared with antibody testing only. 14 The yield has been estimated to be even lower with the currently implemented p24 antigen assay. 14 We therefore conclude that the detection rate of the HIV-1 RNA assay at state public health laboratories most likely is 1 or 2 orders of magnitude higher than the estimated yield of the p24 antigen assay for screening of blood donations. Furthermore, we estimated the cost that would be associated with the implementation of the HIV-1 RNA assay as described herein. The major costs for testing are labor costs and reagent costs. The workload that resulted in the identification of the 2 specimens consisted of 120 determinations for the screening of serum pools and another 40 determinations for the identification of the HIV-1 RNA positive samples from the 2 positive pools. In summary, a total of 80 determinations were required for identification of each HIV- 1 RNA positive sample. In comparison, approximately 100 blood specimens had to be screened with antibody tests for identification of 1 HIV-seropositive blood sample. Most of the HIV-1 RNA assays approved by the US Food and Drug Administration consist of manual procedures and are laborintensive. Furthermore, the generation of serum pools and cataloging of blood samples require considerable time. Ninety-two hours of technologist s time were required for the identification of each HIV-1 RNA positive sample. Therefore, testing of pools with manual HIV-1 RNA PCR assays would not be suitable for large-scale screening of blood samples. However, automation of pooling procedures, sample extraction, and testing for HIV-1 RNA will result in a reduction of labor costs. The reagent cost for the commercially available HIV-1 RNA PCR assay currently is $50 to $90 per assay in the United States, resulting in a cost of $4,000 to $7,200 per previously undetected HIV infection in our tested population. In comparison, total reagent costs for third-generation HIV antibody ELISAs were several times lower, at $389 per HIV-positive blood sample. However, screening of blood donations for p24 antigen has been estimated to result in significantly higher costs per prevented HIV infection than the costs projected for HIV-1 RNA testing in our study population. 15 Additional testing for HIV-1 RNA should be considered for blood samples from high-risk populations, such as the specimens that are submitted to public health laboratories. The higher costs associated with detection of acute HIV infection may be justified for the following reasons: (1) Early diagnosis may be important because of the potential prognostic benefit of early antiretroviral therapy. 16,17 (2) Persons with primary HIV infection are thought to contribute to as much as half of the HIV epidemic, 18,19 so that early identification and treatment of patients with acute HIV infection may be an important step to decrease further spread of the disease. From the 1 Department of Pathology, St Louis University School of Medicine and 2 Pathology and Laboratory Medical Service, VA Medical Center, St Louis, MO; the 3 Missouri Department of Health State Public Health Laboratory, Jefferson City; and the 4 Department of Pediatrics, Washington University School of Medicine, St Louis, MO. Address reprint requests to Dr Ritter: Pathology and Laboratory Medical Service, 113 JC, VA Medical Center, 915 N Grand Blvd, St Louis MO Acknowledgments: We are indebted to Joanne Spadoro, MD, and Ann Butcher, Roche Molecular Systems, Branchburg, NJ, for providing Amplicor HIV-1 Monitor assay kits for this study. We thank James Polarine, Chung Park, Ana Birdwell, Magda Dwidar, and Laura Blair, Washington University School of Medicine, St Louis, MO, for technical assistance with the RNA assays. References 1. Busch MP, Lee LLL, Satten GA, et al. Time course of detection of viral serologic markers preceding human immunodeficiency virus type 1 seroconversion: implication for screening of blood and tissue donors. Transfusion. 1995;35: US Public Health Service. US Health Service guidelines for testing and counseling blood and plasma donors for human immunodeficiency virus type 1 antigen. MMWR Morb Mortal Wkly Rep. 1996;45(RR-2): Mulder J, Resnick R, Saget B, et al. A rapid and simple method for extracting human immunodeficiency virus type 1 RNA from plasma: enhanced sensitivity. J Clin Microbiol. 1997;35: Amplicor HIV-1 Monitor Test Kit [package insert]. Branchburg, NJ: Roche Diagnostic Systems; 1996:7. 5. Ginocchio CC, Wang XP, Kaplan MH, et al. Effects of specimen collection, processing, and storage conditions on stability of human immunodeficiency virus type 1 RNA levels in plasma. J Clin Microbiol. 1997;35: Holmberg SD. The estimated prevalence and incidence of HIV in 96 large US metropolitan areas. Am J Public Health. 1996;86: Gallarda JL, Henrard DR, Liu D, et al. Early detection of antibody to human immunodeficiency virus type 1 by using an antigen conjugate immunoassay correlates with the presence of immunoglobulin M antibody. J Clin Microbiol. 1992;30: Weber B, Moshtaghi-Boronjeni M, Brunner M, et al. Evaluation of the reliability of 6 current anti HIV-1/HIV-2 enzyme immunoassays. J Virol Methods. 1995;55: Constantine NT, van der Groen G, Belsey EM, et al. Sensitivity of HIV-antibody assays determined by seroconversion panels. AIDS. 1994;8: Ritter D, Arens MQ, Walkenbach R, et al. Immunoglobulin M (IgM) sensitive enzyme immunoassays (EIA) detect early seroconversion in HIV infection [abstract]. Clin Chem. 1998;44(suppl):A1140. American Society of Clinical Pathologists Am J Clin Pathol 2000;113:

7 Ritter et al / DIAGNOSIS OF EARLY HIV INFECTION 11. Piatak M, Saag MS, Yang LC et al. High levels of HIV-1 in plasma during all stages of infection determined by competitive PCR. Science. 1993;259: Baumberger C, Kinloch-de-Loes S, Yerly S, et al. High levels of circulating RNA in patients with symptomatic HIV-1 infection. AIDS. 1993, 7(suppl 2):S59-S Morandi PA, Schockmel GA, Yerly S, et al. Detection of human immunodeficiency virus type 1 (HIV-1) RNA in pools of sera negative for antibodies to HIV-1 and HIV-2. J Clin Microbiol. 1998;36: Schreiber GB, Busch MP, Kleinman SH, et al. The risk of transfusion-transmitted viral infections. N Engl J Med. 1996;334: AuBuchon JP, Birkmeyer JD, Busch MP. Cost-effectiveness of expanded human immunodeficiency virus-testing protocols for donated blood. Transfusion. 1997;37: Rosenberg ES, Billingsley JM, Caliendo AM, et al. Vigorous HIV-1 specific CD4 T cell responses associated with control of viremia. Science. 1997;278: Kahn JO, Walker BD. Acute human immunodeficiency virus type 1 infection. N Engl J Med. 1998;339: Cates W Jr, Chesney MA, Cohen MS. Primary HIV infection: a public health opportunity. Am J Public Health. 1997;87: Koopman JS, Jacquez JA, Welch GW, et al. The role of early HIV infection in the spread of HIV through populations. J Acquir Immune Defic Syndr. 1997;14: Am J Clin Pathol 2000;113: American Society of Clinical Pathologists