Routine HIV screening may not detect individuals with primary HIV infection who have yet to form HIV-specific antibodies but who may have high levels of circulating HIV RNA and consequently a high probability of secondary transmission . Pooling sera for RNA testing is feasible and minimizes costs [2–5]. In North Carolina, USA, a study evaluating pooled RNA testing initially reported a 13% increase in HIV detection compared with antibody testing alone  and prompted the evaluation of this approach in King County, Washington, USA.
Most individuals who acquire HIV infection in King County, Washington, USA, are men who have sex with men (MSM). This study evaluated targeted screening for primary HIV infection through pooled RNA testing in MSM.
Public Health - Seattle and King County (PHSKC) uses an HIV test requisition form that records suspected risk factors. Between September 2003 and January 2005, all individuals identified as MSM having confidential or anonymous HIV testing by PHSKC were screened for HIV RNA. The characteristics of this population have recently been described . This study was undertaken to improve the quality of HIV screening and was certified by the University of Washington Human Subjects Division as being exempt from informed consent requirements.
Antibody testing was performed using the Vironostika HIV-1 Microelisa System (bioMerieux, Durham, NC, USA) and was confirmed by Genetic Systems HIV-1 Western blot (Bio-Rad, Redmond, WA, USA). For qualitative pooled RNA testing, sera from 30 antibody-negative individuals were manually combined using an algorithm adapted from Pilcher et al.  for the Procleix HIV-1 Discriminatory Assay (Gen-Probe Inc., San Diego, CA, USA): 200 μl aliquots were combined into three pools of 10 specimens. Aliquots of 500 μl from each of three intermediate pools were combined into one master pool. Pool size was selected to have results available within 2 weeks. With an assay sensitivity of 30 copies/ml, 1: 30 dilutions associated with 30-sample pools permit the detection of samples having 900 copies/ml or greater. Sera from antibody-negative and positive individuals were tested individually to confirm the sensitivity of the pooled assay. If additional serum remained, individuals who were antibody negative using the Vironostika HIV-1 Microelisa System and who had detectable HIV RNA were retested retrospectively using the HIVAB HIV-1/HIV-2 (rDNA) enzyme immunoassay, a third-generation assay (Abbott Laboratories, Abbott Park, IL, USA).
A total of 3525 specimens were screened (Table 1). Eighty-one individuals (2.3%) had detectable HIV antibody and RNA. Seven out of 3439 antibody-negative individuals (0.2%, 95% confidence interval 0.1–0.4%) had HIV RNA by both pooled and individual testing. Three individuals reported symptoms consistent with primary HIV infection; in two cases, primary infection was suspected. Five out of six individuals for whom information was available received results within 3 weeks. HIV infection was confirmed in all seven individuals with suspected primary infection through subsequent testing. Four out of the seven individuals had serum available for retesting using the third-generation HIVAB HIV-1/HIV-2 (rDNA) enzyme immunoassay; two of the four were antibody positive using this assay. None of 3381 available specimens from antibody-negative individuals had detectable RNA when individually tested.
RNA testing increased the yield of HIV antibody testing by 8.6% overall and 6.2% after excluding individuals with suspected primary infection. A total of 204 HIV RNA tests were required to screen 3439 antibody-negative individuals; 29 tests were performed per case of primary infection.
This study supports pooled RNA testing as a practical addition to PHSKC HIV case-finding efforts. RNA testing increased HIV case finding by 6% above a second-generation enzyme-linked immunosorbent assay, similar to the 4% increase observed in more recent evaluation in North Carolina, USA . Relative to other local case-finding methods, finding seven cases are not insignificant. During the study period, PHSKC identified 87 HIV-infected individuals in our sexually transmitted disease clinic and 15 individuals through three bathhouse-based testing sites.
Although pooled testing was 100% sensitive in our study compared with individual testing, pooling may dilute samples with low-level viremia below the limit of detection. Individuals with primary HIV infection typically have high-level viremia , but HIV transmission has been documented via blood donations from individuals with primary infection screened by pooled RNA testing [9,10]. RNA testing of individual samples would increase the sensitivity of HIV screening, but it would do so at a high incremental cost per case identified.
This study is distinct from previous studies in that screening for primary infection was targeted to a population with the highest HIV incidence. The utility of targeted screening in other settings will vary based on local HIV epidemiology and ease of identification. Furthermore, the incremental benefit of RNA screening could be reduced by continued advances in the sensitivity of antibody testing (as at least two of the individuals diagnosed in our study by pooled RNA testing would have been identified as HIV infected by the use of a third-generation enzyme immunoassay) or by an increase in the clinical recognition of the acute HIV syndrome. Some patients seek attention at public health clinics for symptoms associated with primary infection, and health departments, regardless of the use of pooled RNA testing for screening, should have RNA testing available to diagnose suspected primary infection.
The cost effectiveness of universal HIV antibody screening has recently been evaluated [11,12]. However, depending on the proportion of screening that would occur through public health testing sites, the actual costs required to screen entire populations may be prohibitive for local health departments, and targeted screening may identify more individuals at a lower cost. Under current circumstances, we believe that targeted screening for primary HIV infection through pooled RNA testing in MSM is a useful addition to HIV case-finding efforts.
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