Objective

To develop a pooling method for detection of viral RNA for diagnosis of acute HIV infection and estimation of HIV-1 incidence.

Methods

Sera from 700 consecutive seronegative patients attending sexually transmitted disease clinics in Pune, India, were screened individually for p24 antigen, and pooled into seven pools of 100 for detection of HIV-1 RNA by reverse transcriptase-polymerase chain reaction. HIV-1 incidence was calculated by the traditional cohort method, the p24 antigen method, and a multistage pooling method in which RNA-positive pools were re-analyzed in smaller pools.

Results

Sera from 700 individuals were grouped into seven pools of 100, of which four were positive. These four positive pools were subdivided into eight pools of 50, of which seven were positive. The seven positive pools were subdivided into 35 pools of 10, of which 10 were positive. Based on the 10 RNA-positive pools, the point estimate of HIV-1 incidence was 19.9% per year [95% confidence interval (CI), 7.3–31.8%]. Of the 700 samples analyzed for p24 antigen, eight were positive, resulting in a point estimate of incidence of 18.5%/year (8.0–36.5%). In contrast, the incidence rate based on the traditional cohort method of follow-up was lower at 9.4%/year (4.8–16.4%) due to a low follow-up rate. Testing of individual samples from the 10 RNA-positive pools identified 10 individuals with acute primary HIV-1.

Conclusion

The multistage pooling method for detection of HIV-1 RNA was more sensitive than the p24 antigen method, and was five-fold less expensive than the p24 antigen assays. Pooling samples for RNA detection was effective in estimating current incidence rates with cost savings that would be practical for use in developing countries.