Longitudinal Variability of Human Immunodeficiency Virus Type 1 RNA Viral Load Measurements by Nucleic Acid Sequence-Based Amplification and NucliSens Assays in a Large Multicenter Study

Assay format and input volume.Table1 shows overall means and standard deviations (SD) of the log-transformed results of the external standards for each assay-input volume combination. Observed variability was lowest for NucliSens_{1.0 ml} and was similar for NASBA_{0.1 ml}.

In a retrospective analysis of assay run acceptability, we defined run rejection criteria as (i) two results exceeding the target value by more than 2 SD, (ii) one result exceeding the target value by more than 3 SD, or (iii) an R square of <0.95 (8). Overall rejection rates were 6% for NASBA_{0.1 ml}, 3% for NucliSens_{0.2 ml}, and <1% for NucliSens_{1.0 ml}(data not shown).

Fluctuations in HIV concentrations greater than 0.5 log₁₀unit have been described as clinically relevant (7, 10, 12, 13). The NASBA_{0.1 ml} result was more than ± 0.5 log unit from the target of 10^4.18 copies/ml for 23 of 474 runs (5%) compared to 0 of 178 runs for NucliSens_{0.2 ml} and 0 of 162 runs for NucliSens_{1.0 ml}(P < 0.0001) . NucliSens_{0.2 ml} missed the target of 10^3.18 copies/ml 26 times (15%) compared to 0 times for NucliSens_{1.0 ml} (P < 0.0001) .

Laboratory differences.There were statistically significant differences in levels of variance between labs for the lowest-range standard of all three assay-input volume combinations and for the highest-range standard for NucliSens_{1.0 ml} (data not shown).

Variability over time.Table 2summarizes SD for 6-month time intervals corresponding to WIHS visit windows for each assay-input volume combination and VQA external standard. Within each combination and for all standards, the SD was stable and there were no statistically significant trends except with the lowest-range control value for NucliSens_{0.2 ml}.

For NASBA_{0.1 ml} (April 1996 to October 1998), the observed nonlinear trends were statistically significant for all standards (lowest range, P = 0.003 [Fig.1A]; middle range, P < 0.0001 [Fig. 1B]; highest range, P < 0.0001 [Fig. 1C]). NucliSens_{0.2 ml} (October 1997 to January 1999) demonstrated a marginally significant trend in the highest-range standard (Fig. 1F; P = 0.03 ). NucliSens_{1.0 ml} (February 1999 to January 2000) demonstrated significant trends in the middle and high-range standards (Fig. 1H, P = 0.003 , and I, P = 0.001 , respectively).

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Fig. 1.

Scatter plots with nonlinear smooth curves of all log-transformed results over the observation period of each assay for each of the three VQA controls (solid line). Each dot represents the result from one run. Nominal target values (dashed lines) and values 2 SD above and below the nominal value (dotted lines) give context to the measurement fluctuations over time. NonlinearP values indicate the strength (fit) of the association between time and the control result. (C) 1.5 × 10⁶copies/ml; (B, F, and I) 1.5 × 10⁵ copies/ml; (A, E, and H) 1.5 × 10⁴ copies/ml; (D and G) 1.5 × 10³ copies/ml.

While we observed no gain in precision in the transition from NASBA_{0.1 ml} to Nuclisens_{0.2 ml}, there was a gain in precision and reproducibility for all standards in the transition from Nuclisens_{0.2 ml} to Nuclisens_{1.0 ml}. This was most likely the cumulative result of kit reformulation and increased input volume (M. Cronin, OTC, personal communication, 2000; Introducing a New Family of Assays Based on NASBA, OTC pamphlet, 1997; NucliSens: Synergy of Strengths, OTC pamphlet, 1997).

Theoretically, the observed systematic decrease in results over the 2.5-year observation period for NASBA_{0.1 ml} may be an indication of the deterioration of the virus in the viral stock used for VQA standards over time. However, an internal VQA study to address this possibility found no indication of deterioration of the virus in quality assurance panels (3). The observed increase in the level of HIV-1 RNA over the 1-year observation period for NucliSens_{1.0 ml} also argues against deterioration of the virus in the viral stock. All statistically significant longitudinal trends were well within 2 SD, indicating no biologically relevant bias. We conclude that it is not necessary to make an intra-assay adjustment for time.

We have described the performance over a period spanning the introduction of HAART of three versions of OTC assays within an externally controlled multilaboratory program. NASBA technology is invariant to specimen type, an advantage that makes this assay valuable to clinicians and researchers testing nonplasma body fluids for HIV-1 RNA. Our data demonstrate that the precision of the OTC assays is comparable to that of other assays not invariant to specimen type. We also demonstrated that patient viral load levels in the HAART era can be measured with the same precision as or with better precision than in the pre-HAART era, despite an average HIV-1 RNA decrease of 2 log₁₀ units. We have shown that HIV-1 RNA measurements across multiple WIHS sites over a period of 4 years were comparable despite the introduction of HAART, different versions of the assay, and differences between labs.