Results

The sensitivity of the RT-PCR procedure was first tested using the HIV1 cRNA standards.Different concentrations of HIV1 cRNA（1 to 10，000 molecules per test）were reverse-transcribed into cDNA using RT，followed by PCR.Using this procedure，the lower limit of detection of HIV1 RNA was 10 copies and the linear range of detection was between 50 and 1，000 copies.We also found that identical quantitative data were obtained for the standards when they were amplified alone or in the presence of plasma extract.Under the acidic conditions of the RNAzol extraction method，RNA is isolated exclusively，and contaminating DNA was not detected in our samples.The loss of RNA in the extraction step was small，with yields consistently greater than 80%.The intra-assay coefficient of variation was found to be 16%（Zhu et al.，1995）.

Figure 1 illustrates the analysis of HIV1 RNA gag sequences from 30 of the 53 subjects.The patient population included 2 seronegative individuals and 28 seropositive patients diagnosed as asymptomatic，ARC or AIDS.Duplicate plasma samples were prepared and analysed for each individual，and the duplicates consistently yielded similar results with less than 2-fold variation between samples.While one HIVl-seronegative individual（No.29）was negative for HIV1 RNA even when the autoradiograph was prepared by overnight exposure，the other HIV1-seronegative individual（No.7）was positive for HIV1 RNA by RT-PCR（7，000 copies/ml）.This high-risk subject is additionally HTLV-I-seropositive and is the active sexual partner of an HIV-seropositive individual.All 28 seropositive patients had positive RT-PCR results for HlV1 RNA.The HIV1 RNA copy numbers measured by RTPCR for the seropositive patients varied from 800 copies/ml to 500，000 copies/ml.As illustrated in Figure 1，the band intensities of HIV1 RNA from six patients（Nos.3，4，10，18，22 and 24）were out of the RT-PCR quantitative range.These plasma samples were further diluted to the proper concentrations so that accurate quantitation could be determined.

Figure 1　Autoradiographs of HIV1 RNA PCR products obtained from plasma of 30 patients

HIV1 RNA was prepared as described in“Patients and Methods”.Samples were run in duplicate

Additional testing（data not shown）of another 10 HIV1-seropositive patients and 13 HIV1 seronegative individuals yielded similar results.All 10 seropositive patients were found to be positive for HIV1 RNA by RT-PCR.Conversely，all 13 HIVl-seronegative individuals were shown to be negative for HIV1 RNA after overnight autoradiograph exposure.However，in one high-risk seronegative individual with a history of multiple blood transfusions and hepatitis B virus infection，the RT-PCR assay yielded an extremely faint signal.Follow-up of this patient is continuing at present.

In order to investigate the relationship between plasma HIV1 RNA concentration and disease status，HIV1 RNA copy numbers were plotted against CD4+T-cell counts.The results are shown in Figure 2（a）.Overall，asymptomatic and AIDS patients showed a good correlation between HIV1 RNA copies and CD4+T-cell counts，while ARC patients were more variable.An increase in HIV1 RNA copies correlated with a decrease in CD4+T-cell counts in all AIDS patients except for nos.16，23 and 28 from Figure 1.In general，ARC and AIDS patients had higher HIV1 RNA levels compared with asymptomatic individuals（usually above 25，000 copies/ml）.HIV1 RNA was also measured in serial plasma samples from five patients over 1—3 years，as shown in Figure 2（b）.Three patients had ARC，while two patients had AIDS.All patients were on stable doses of AZT at the time of plasma sampling，and one patient（MC）was also taking ddC.In two of the ARC patients（DE and RA），a rise in CD4+T-cell counts was associated with a greater than 2-fold fall in HIV1 RNA copies.Conversely，in the AIDS patients（MO and MC），a decline in CD4+T-cells corresponded to a greater than 2-fold rise in HIV1 RNA.The third ARC patient（YE）had fluctuating levels of CD4+T-cells that corresponded to fluctuating HIV1 RNA levels.(https://www.daowen.com)

The relationship between HIVI RNA levels and AZT treatment is shown in Table 1.The mean HIV1 RNA level in AZT-treated patients was 64，174 copies/ml，while the mean level in patients on no therapy was 17，200 copies/ml.These levels parallel the CD4+T-cell counts shown in Figure 2（a），with several exceptions（see below）.

Figure 2　（a）Correlation between plasma HIV1 RNA levels（copies/ml），CD4+T-cell counts（cells/mm3）and disease status in HIV1-seropositive patients Asympt=asymptomatic HIV1 seropositive；ARC，AIDS-related complex
（b）HIV1 RNA levels in serial plasma samples from patients with ARC and AIDS.Time points represent 3—6-month intervals