Bhupat D. Rawal

Use of laboratory tests and clinical symptoms for identification of primary HIV infection

Objective To determine the sensitivity and specificity of symptoms, three HIV-1 RNA assays, a p24 antigen EIA and a third-generation enzyme immunoassay (EIA) antibody test for diagnosis of primary HIV infection (PHI). Design Prospective cohort in a university research program. Participants Of 258 eligible persons screened for PHI, 40 had primary/early infection (22 preseroconversion, 18 within 6 months of seroconversion) and 218 did not. Seven participants with preseroconversion HIV-1 from a second center were added for evaluating laboratory tests. Main outcome measure PHI, defined as a negative or indeterminate antibody test with subsequent conversion. Symptom analysis also included persons with antibody conversion of less than 6 months’ duration. Results The symptoms most strongly associated with PHI in multivariate analysis were fever [odds ratio (OR) 5.2; 95% confidence interval (CI) 2.3–11.7] and rash (OR 4.8; 95% CI 2.4–9.8). The sensitivity and specificity, respectively, for detecting preseroconversion HIV infection were: p24 antigen, 79% and 99%; third-generation EIA, 79% and 97%; HIV-1 RNA by branched chain DNA 100% and 95%; HIV-1 RNA by polymerase chain reaction 100% and 97%; HIV-1 RNA by transcription-mediated amplification testing, 100% and 98%. False-positive HIV-1 RNA tests were not reproducible and had values < 3000 copies/ml, while only one person with confirmed PHI was in this range. Conclusions Rash and fever indicated the highest risk of PHI. HIV-1 RNA tests are very sensitive for PHI but false-positive results occur. False-positive results can be reduced through duplicate testing and considering tests < 5000 copies/ml as indeterminate results requiring additional testing. p24 antigen was more specific than HIV-1 RNA testing but less sensitive.

Lack of correlation between HBsAg and HBV DNA levels in blood donors who test positive for HBsAg and anti-HBc: implications for future HBV screening policy

BACKGROUND:  Studies showing a significant correlation between hepatitis B surface antigen (HBsAg) and hepatitis B virus (HBV) deoxyribonucleic acid (DNA) levels have focused on the HBV seroconversion window period.

Detection of early HIV infection and estimation of incidence using a sensitive/less-sensitive enzyme immunoassay testing strategy at anonymous counseling and testing sites in San Francisco.

Timely estimates of HIV incidence are needed to monitor the epidemic and target primary prevention but have been difficult to obtain. We applied a sensitive/ less-sensitive (S/LS) enzyme immunoassay (EIA) testing strategy to stored HIV-positive sera (N = 452) to identify early infections, estimate incidence, and characterize correlates of recent seroconversion among persons seeking anonymous HIV testing in San Francisco from 1996 to 1998 (N = 21,292). Sera positive on a sensitive EIA but negative on a less-sensitive EIA were classified as early HIV infections; sera positive on both EIA were classified as long standing. Seventy-nine sera were from people with early HIV infection. Estimated HIV incidence was 1.1% per year (95% confidence interval [CI], 0.68%-1.6%) overall and 1.9% per year (95% CI, 1.2%-3.0%) among men who have sex with men (MSM). Early HIV infection among MSM was associated with injection drug use, unprotected receptive anal sex, and multiple sex partners in the previous year. No temporal trend in HIV incidence was noted over the study period. The S/LS strategy provides a practical public health tool to identify early HIV infection and estimate HIV incidence in a variety of study designs and settings.

Reduction of human immunodeficiency virus- infected cells from donor blood by leukocyte filtration

Several filters for leukocyte removal were evaluated in terms of their ability to reduce the cell‐associated human immunodeficiency virus (HIV) load in units of blood either inoculated in vitro with lymphocytes from a chronically infected cell line or collected directly from seropositive donors. Filtration of the experimentally inoculated units of blood resulted in a 5.9 log10 mean reduction (95% confidence interval:7.4–4.5) of tissue culture infectious units (TCIU) as assayed by end‐point titration using the cocculture assay. Filtration of the units of blood from anti‐HIV positive donors lowered the infectivity by over 2 logs, as detected by the coculture and polymerase chain reaction (PCR) techniques. However, residual cell‐associated virus was detected in the majority of experiments. Clinical studies are warranted to determine if leukocyte filtration of blood will reduce the risk of transfusion transmitted viral infections.

Risk factors for incident HIV infection among anonymous HIV testing site clients in Santos, Brazil: 1996-1999.

Objectives To determine temporal trends in HIV infection and risk factors among persons seeking anonymous HIV testing in Santos, Brazil. Methods Data and sera from persons testing for HIV from 1996 to 1999 were used. Exposures were abstracted from HIV testing risk assessments. Stored HIV-positive sera were tested to identify recently acquired HIV infection using a serologic testing algorithm for detecting recent HIV seroconversion (STARHS). Independent associations between exposures and recently acquired HIV infection were determined using multivariate analyses. Results Overall, estimated HIV incidence was 2.0% (95% CI: 1.1–3.5) for the 4-year period: 1.2% (95% CI: 0.5–2.6) in women and 2.7% (95% CI: 1.3–5.0) in men. Incidence increased among women but remained stable among men. Exposures independently associated with incident infection included a history of sex work (OR= 5.4, 95% CI: 1.5–18.7), concurrent syphilis infection (OR =4.1, 95% CI: 1.4–11.9), anal sex (OR = 3.0, 95% CI: 1.3–7.1), and having an HIV-positive sexual partner (OR= 1.4, 95% CI: 1.1–1.9). Conclusions This study further demonstrates the public health utility of using the STARHS for the assessment of emerging trends in the HIV epidemic. Results from this study will help to target appropriate prevention strategies directed toward at-risk populations in Santos.

Use of the sensitive/less-sensitive (detuned) EIA strategy for targeting genetic analysis of HIV-1 to recently infected blood donors

Objective To corroborate the validity of the recently developed sensitive/less sensitive (S/LS) dual enzyme immunoassay (EIA) strategy for the detection of recently infected individuals and to genetically analyze recently transmitted strains of HIV-1 in a US blood donor population. Design The S/LS EIA strategy was used to identify 33 recently infected subjects among 281 enrolled HIV-1 seropositive blood donors (from a total of 410 HIV-1 infected subjects identified from 5 230 463 blood donations screened by participating US blood centers in 1995–1996). Methods We analysed three host response and viral characteristics were associated with recent HIV-1 infection: rapidly increasing EIA optical density (OD) values, genetically homogeneous env gene quasispecies, and putative non-syncytium inducing env V3 loop sequences. The drug resistance genotypes of the recently transmitted strains were determined by DNA sequencing. Results Increasing EIA OD values, clonal HIV-1 quasispecies and V3 loop sequences with inferred NSI phenotypes were generally detected in LS EIA non-reactive samples. Thirty-two subtype B and one CRF02_AG recombinant HIV-1 were detected. Genetic evidence for drug resistance to zidovudine (K70R) and non-nucleoside analog reverse transcriptase inhibitors (V108I) was detected in one strain each, and three other strains showed the presence of accessory protease inhibitor resistance mutations. Conclusions Immunologic and virologic results further substantiate the validity of the S/LS EIA strategy for the detection of recent infections and illustrate its use for targeting molecular and epidemiological investigations to incident cases identified from large cross-sectional screening programs, rather than the more costly and logistically difficult longitudinal studies.

Leukocyte filtration removes infectious particulate debris but not free virus derived from experimentally lysed HIV-infected cells

Abstract. We used in vitro model of storage‐induced leukocyte degradation in blood to experimentally characterize the infectivity of the debris of lymphocytes harboring human immunodeficiency virus (HIV‐1). The leukocyte filtration process removed both intact HIV‐infected H9 cells and the particulate debris, but failed to remove the cell‐free HIV released from lysed cells. These data suggest that the leukocyte filtration of donor blood soon after collection would optimally reduce the particulate infectious burden of blood for transfusions.

Rare detection of hepatitis B and hepatitis C virus genomes by polymerase chain reaction in seronegative donors with elevated alanine aminotransferase

Background: Since screening for antibody to hepatitis C virus (HCV) was introduced in 1990, posttransfusion hepatitis has been reduced to nearly background levels. This has led to reconsideration of the value of testing donated blood for elevated alanine aminotransferase (ALT). The contribution of ALT testing in detecting seronegative infection was evaluated by the performance of polymerase chain reaction (PCR) for hepatitis B virus (HBV) or HCV in plasma from ALT‐elevated blood units.

Complement Killing of Yersinia enterocolitica and Retention of the Bacteria by Leucocyte Removal Filters

We report studies on the complement sensitivity of four strains of Yersinia enterocolitica, serotypes O:3, O:9, O:5,27, and O:20, isolated from blood units involved in transfusion fatalities. Complement in fresh CPD plasma killed Y. enterocolitica within 4 h at 22°C in 100% of the experiments. The bactericidal action was serotype and complement activation pathway dependent. Both classic and alternate pathways seemed to be active, but the latter to a lesser degree. When the classic pathway was blocked by chelation of Ca2+ no complete killing was obtained. Complement did not enhance or condition Yersinia for leucocyte filter retention. Direct removal of Yersinia by filtration was also related to serotype; all strains were reduced by filtration in heat‐inactivated plasma, and all except serotype O:5,27 were reduced in Ca2+ ‐chelated plasma. Our findings may explain why plasma products and platelet concentrates are rarely involved in Yersinia sepsis related to transfusion.

In vitro inactivation of human immunodeficiency virus by ascorbic acid

In vitro inactivation of cell-free human immunodeficiency virus (CFHIV) was investigated by mixing replication-competent virions with aliquots of a culture medium (RPMI) containing increasing amounts (62.5-500 micrograms/ml) of ascorbic acid (AA) at pH7. Similarly, mixtures of CFHIV and 500 micrograms/ml AA in whole blood (WB) and leukocyte depleted blood (LDB) were made; control mixtures containing either CFHIV or AA alone in each experiment were included. After holding the mixtures for 3 h at 4 degrees C, the tubes containing WB and LDB mixtures were centrifuged to remove the blood cells. The respective supernatants, including the control aliquots, were layered over 0.5 x 10(6) MT2 cells in quadruplicate wells in microtitre plates. After 1 h of incubation at 37 degrees C in an atmosphere of 5.0% carbon dioxide to permit contact of viable virions, the fluid in each well was replaced with RPMI containing 20% fetal bovine serum (FBS). The incubation was then continued at 37 degrees C for 5 days. On the basis of (1) absence of syncytia formation, (2) 100% viability of MT2 cells as compared with the cell controls, (3) absence of p24 antigen in the culture supernates, and (4) absence of HIV DNA in MT2 cells, we conclude that 500 micrograms/ml AA, in (a) RPMI, (b) WB, or (c) LDB, inactivated CFHIV in vitro. Furthermore, we determined that addition of 500 micrograms/ml AA to platelet concentrates did not adversely affect the platelet function tests during 5 days of storage at room temperature. These data warrant further work to evaluate the mechanism of CFHIV inactivation by treatment of blood products with AA.