Quantitation of human immunodeficiency virus plasma RNA by branched DNA and reverse transcription coupled polymerase chain reaction assay methods: A critical evaluation of accuracy and reproducibility

J Todd, T Yeghiazarian, B Hoo, J Detmer, J Kolberg, R White, J Wilber, M Urdea

Nucleic Acid Systems, Chiron Corporation, Emeryville CA 94608-2916, USA

Summary

The present study was designed to evaluate the utility of two assays, reverse transcrip- tion coupled polymerase chain reaction (RT-PCR) and branched DNA (bDNA), to accurately and reproducibly quantitate plasma human immunodeficiency virus (HIV) RNA levels. The bDNA assay quantitated RNA transcripts, prepared from different HIV-1 subtypes (A-E), within l.&fold. Similarly, the bDNA assay, standardized to subtype B, was used to quantitate cultured isolates from subtypes A, C-F within 2-fold; however, the RT-PCR assay displayed a 904-fold range. Reproducibility studies demonstrated that the bDNA and RT-PCR assays could be used statistically (PcO.05) to discern less than 2- and 6.8-8.1-fold changes in RNA levels, respectively. This study showed that the two assay methods differ in accuracy and reproducibility. These differences need to be considered when choosing specific applications for the methods.

Key words: PCR, HIV, branched DNA, quantitation, RNA

Serodiagn. Immunother. Infect. Disease 1994, Vol. 6, 233-239, December

Introduction

Quantitation of viral genomic nucleic acids, either RNA or DNA, has become an effective means for determining the level of specific viruses in clinical speci- mens”“. In chronically infected individuals, these nucleic acids are generally present in low concentra- tions and the methods used for quantitation usually employ some form of amplification process. Many of the methods, such as polymerase chain reaction (PCR)3J4, reverse transcription coupled to PCR (RT- PCR)2.4Js, quantitative competitive PCR (QC- PCR)sJ”“, immune capture RT-PCRi8, and nucleic acid sequence based amplification (NASBA)i9 are based upon enzymatic amplification of target sequences. In

Received: 3 August 1994 Reviewed: 8 September 1994 Accepted: 20 September 1994 Correspondence and reprint requests to: J Todd, Nucleic Acid Systems, Chiron Corporation, 4.560 Horton Street, Emeryville CA 94608.2916, USA 0 1994 Butterworth-Heinemann Ltd 0888-0786/941040233-07

contrast, our laboratory chose to focus on signal ampli- fication and has developed quantitative virological assays based on branched DNA (bDNA) technol- ogyi1-i3Jo. This methodology allows for the direct measurement of nucleic acids at their naturally occur- ring levels and circumvents some of the errors inherent in extraction and amplification of target sequences.

Reproducibility for viral quantitation will be of paramount concern to clinicians. Although lo-fold changes in human immunodeficiency virus (HIV) RNA levels in plasma have sometimes been noticed upon administration of antiviral drugs such as AZT’AS.“, more subtle changes1,4.sJ1.20 are often observed and could be meaningful. Most studies to date have employed the so-called ‘batch mode’ testing approach; that is, all samples for one study are run at the same time with one operator and one lot of reagents. In contrast, in a clinical setting, it is likely that samples will be tested in real-time by different operators on different days (months and years) with different lots of reagents. To understand the quantita- tive results generated from the latter type of testing, a

234 Serodiagn. Immunother. Infect. Disease 1994; 6: No 4

thorough understanding of assay reproducibility is required. In addition, the accuracy of HIV RNA assays and nucleic acid quantitation is likely to become increasingly significant. For instance, Lau et al.9 reported that a low serum level of hepatitis C virus (HCV) RNA prior to therapeutic administration of interferon-o (Y was the best prognostic indicator of long term response. Evidence is mounting that HIV RNA levels have prognostic value. The time frame for disease progression varies widely in individuals infected with HIV in that progression to AIDS can proceed within 4 months of infection or can take longer than 12 yr, if at alp-23. Since HIV infection results in the presence of detectable virus in the plasma of virtually all patients5, the presence of virus alone cannot predict progression. In contrast, recent reports have shown that there is a strong and statist& tally significant relationship between the level of HIV RNA in blood (plasma and peripheral blood mononu- clear cells) and disease progression24-26. Mellors et al.24.2s followed a cohort of HIV-infected patients with known dates of seroconversion and demonstrated, using the bDNA assay, that in subjects with plasma HIV RNA levels less than lo4 equivalents ml-’ plasma (Eq ml-‘) for the first 2 yr after seroconversion, the proportion developing AIDS within 5 yr was ~0.06. In contrast, when more than one plasma specimen provided RNA quantitation greater than 104 Eq ml-’ within the first 2 yr of seroconversion, the proportion developing AIDS ranged from 0.45-0.86. Therefore, since a relationship appears to exist between HIV plasma RNA levels and clinical progression, it is important for this measurement to be made in an accurate manner.

A number of different target and signal amplifica- tion assays have been developed for the quantitation of HIV RNA in plasma. In general, these assays have yielded comparable results, demonstrating that HIV RNA can be detected in most if not all infected patients2,4JJ1,1s, that HIV RNA levels are in general inversely correlated with CD4 cell counts4JJ’ and that decreases in HIV RNA can be shown to be associated with intervention with HIV anti-viral therapy2JJ1J7. The Quantitative Virology Working Group of the NIH/NIAID AIDS Clinical Trials Groups (ACTG) has recently completed a multicentre evaluation of six different quantitative HIV plasma RNA assay methods, including bDNA and RT-PCR and concluded that several of the assays are ready for use in clinical trialszx. Although these assays appear to provide promise for quantitating viral burden via measurements of RNA levels, there is a lack of detailed understanding concerning the accuracy and reproducibility of the methods.

The present study was designed to determine the reproducibility of bDNA and RT-PCR assays and understand its impact on the ability to determine changes in HIV RNA levels over the course of therapy intervention and disease progression. The second aspect of the study was to assess the impact of HIV

RNA genotype variation on the accuracy of quantita- tive plasma HIV RNA measurements made with these two methods.

Methods

Quantitative HIV RNA assays

The bDNA assay has been described in detail elsewhere”. In summary, virus is concentrated from plasma by centrifugation (23 500 X g, 1 h) and then lysed with proteinase k and detergents to release RNA. Genomic RNA is captured onto the surface of a well in a 96-microwell plate. Through a series of hybridiza- tion events, multiple branched DNA (bDNA) and alkaline phosphatase-labelled oligonucleotide mole- cules are bound to the immobilized target nucleic acid. Enzyme-triggerable dioxetane substrate is added, and the resultant steady state chemiluminescent signal, which is proportional to the amount of nucleic acid present, is measured in a luminometer. Quantitation of unknown specimens is determined by interpolation using a standard curve and results are given as equiv- alents ml-r (Eq ml-l). An RNA equivalent is the relative luminescence corresponding to one copy of a HIv-11llt3 RNA standard. The bDNA assay was performed at Chiron Corporation (Emeryville, CA, USA). The RT-PCR method has been described elsewhereIs. This method differs from other RT-PCR methods in that an internal RNA standard is used to normalize for inhibition of the RT-PCR reactions. After the reverse transcription and PCR reactions which use biotinylated-oligonucleotides, amplified products are captured in microtitre wells and detected using avidin conjugated horseradish peroxidase and a chromogenic substrate mixture. The RT-PCR assay was performed at Roche Biomedical Laboratories, Inc. (Research Triangle Park, NC, USA) by trained person- nel.

Specimens

HIV positive plasma (antibody, p24 antigen and Western blot reactive) were obtained from Serologicals Inc. (Murietta, GA, USA). HIV negative plasma was obtained from North American Biologicals Inc. (Miami, FL, USA). An HIV panel was prepared by making dilutions of positive plasma into the negative plasma. One ml aliquots were prepared and stored frozen at -70” C until tested by RT-PCR or bDNA assays. Virus isolates (culture supernatants) represent- ing subtypes A, C, D, E and F were obtained from John Mascola MD at the Walter Reed Army Institute of Research (Rockville, MD, USA). Subtype B (HIV%?!) has been previously describedz9. The subtype categories were determined by sequence homology analysis of the env region of the genome”“. The isolates were thawed and used for cloning (see below) or diluted into HIV

Todd et al.: Quantitation of HIV plasma by branched DNA and RT-PCR assay methods 235

negative plasma. aliquoted, and frozen at -70” C, until tested by bDNA and RT-PCR assays. Specimens that were tested by RT-PCR were shipped frozen to Roche Biomedical Laboratories on dry ice. All specimens were coded and tested by either bDNA or RT-PCR in a blinded fashion.

and variance equal to u?. Given these assumptions, the random error variance was estimated from an ANOVA. This analysis was done using the VARCOMP procedure in SAS vs. 6.07.

Results

HIV RNA preparations Reproducibility of bDNA assay

A 2829 bp pol fragment of each subtype (nucleotide positions 2352-5181) was prepared by RT-PCR ampli- fication of purified RNAjJ. This fragment covered the region in the pol gene that is recognized by all 49 of the target specific probes in the bDNA assay”. Sequence homology to subtype B ranged from 89-92%. In vitro transcripts were prepared and the transcribed RNA was quantified by three methods, phosphate analysis using the NIST standard, hyper- chromicity and optical density at A26,j 31-.13. Serial dilutions of the HIV RNA transcripts ranging from 6 x lob-1.2 x 1Oj molecules ml-’ plasma (proteinase k treated) were tested in the bDNA assay over six assay runs.

Statistical analysis

The within-assay. within-operator and overall variance components for different reagent lots of the bDNA assay were estimated using the following linear model:

log(Eqs ml-‘) = p + operator, + day(operator),,,, + E;,~

where operator, is the effect due to operator, day(operator),,j, is the effect due to day (which is nested in operator) and E,~~ is the within-assay random error. This assumes that the log(Eqs ml-‘) were normally distributed and the E;,, were independently, identically and normally distributed with mean equal to zero and variance equal to I+. The variance compo- nents were then estimated from an analysis of variance (ANOVA). This analysis was done using nested proce- dure in SAS vs. 6.07 (SAS, Research Triangle Park, NC, USA). The ANOVA for RT-PCR was performed by first separating the samples into two groups: those detected by both assays and those detected only by the RT-PCR assay. Certain assumptions were made in estimating the random error variance: first, since it is unknown which samples were run together on a plate (which would be the case when clinical results are reported back to the clinician), it is assumed that each determination was independent, i.e. that there were no plate/sample interactions. This resulted in the follow- ing linear model:

Reproducibility of RNA quantitation using the bDNA assay was determined by testing replicates of a single specimen over 94 unique assay runs. Quantitation was obtained from the mean of two determinations”, and since there were quadruplicate determinations per assay run, two independent quantitations of the speci- men were obtained per assay run. The mean value for all quantitations (66 X 103 RNA Eq ml-l) was located in the lower 5% of the standard curve. where the great- est assay variability would be expected. As shown in Table 1, panel A, the coefficients of variation (CV), ranged from 7-lo%, for within-assay and 18-23% overall. The within-assay variability represents that which one would find when specimens were tested in a ‘batch mode’, whereas the overall variability repre- sents error due to within-run as well as between opera- tors on different days. Of particular interest was the fact that the variability did not greatly change through three separate lots of reagents. The random error variances were estimated and are shown in Table 1, panel B. These estimates were used to predict the upper 95% tolerance limit for the difference between two log quantitations and are presented in Table 1. panel C. A fold difference greater than this number would have less than a 5% chance of happening by random. These data indicate that if two sequential specimens from the same patient were being analysed for changes in RNA levels, when tested in a batch mode, a 1.30-1.41-fold change would be statistically significant (PcO.05). In a similar fashion, if the speci- mens were tested separately, on different days by different operators. a 1.78-1.99-fold change in RNA levels would be required to be significant at the PcO.05 level.

Reproducibility of bDNA and RT-PCR

log copies ml-’ = 11 + sample, + e,

where u is the grand mean, sample, is the sample effect and e, random error. The second assumption was that the log copies ml- I and e,‘s were independently, identi- cally and normally distributed with mean equal to zero

A comparison of RNA quantitation by the bDNA and RT-PCR assays was made by testing an identical plasma panel over three separate assay runs by each assay method. A summary of the results are presented in Table 2. The inter-assay coefficient of variations ranged from 1342% for the bDNA assay and 43-93% for the RT-PCR assay. The %CV did not increase or decrease in a consistent manner throughout the dilution series. indicating that the variability is not influenced by the level of RNA in the specimen. Of particular interest was the fact that only panel member C displayed bDNA assay variability which was larger than that noted above.

236 Serodiagn. Immunother. Infect. Disease 1994; 6: No 4

Table 1. Reproducibility of HIV RNA quantitation using the bDNA assay*

A Kit lot No. of Coefficient of variation

operators Overall Within- Within-assay

operator % % %

1 5 18 18 10 2 12 20 17 10 3 4 23 10 7

B Kit lot Variance estimates+

Overall Within-operator Within-assay

1 0.0303 0.0206 0.0097 2 0.0387 0.0217 0.0121 3 0.0413 0.0053 0.0052

C Kit lot Significant fold change iP<O.O5)

Overall Within-operator Within-assay

1 1.78 1.79 1.42 2 1.91 1.88 1.49 3 1.99 1.42 1.30

‘Calculations were based on the results from 94 assay runs. tEstimates were based on log Eqs ml-‘.

Table 2. Determination of reproducibility of quantitation for bDNA and RT-PCR

Method ID Quantitation mP* (X 103)

Run 1 Run 2 Run 3

Fold range t

CV %

RT-PCR A 206 ND B 213 54 C 99 ND D 29 7.2 E 14 ND F 6.5 4.9

G 4.4 2.1 H 3.4 1.1 I 1.3 0.5

bDNA A 58 56 El 32 38 C 10 17 D 8 12 E <5 7 F <5 5

G <5 <5

95 41 33 10

6.5 2.5 1.3 0.8 0.3

42 30

7 9 6

15 <5

2.2 52 5.2 93 3.0 71 4.1 76 2.2 48 2.6 43 3.4 62 4.3 82 4.3 74

1.4 17 1.3 13 2.4 42 1.5 20 1.2 13

ND ND ND ND

‘Quantitation expressed as RNA copies ml-’ for RT-PCR and RNA Equivalents ml-’ for bDNA. tHighest quantitation/lowest quantitation for a given specimen. ND, Determination not made.

This anomaly may be due to a smaller sample size or due to variability associated with the plasma sample itself. Consistent with RT-PCR displaying higher %CVs, the RT-PCR assay produced a larger fold range in quantitation (2.2-5.2) compared to the bDNA assay (1.2-2.4). The largest fold variation obtained with the bDNA assay was comparable to the smallest fold varia- tion obtained with the RT-PCR assay. The differences

in quantitations between the two assays were consistent with the RT-PCR providing values that were 1.6 to 3.1 times greater. Since the panel consisted of the same positive specimen diluted to various final concentra- tions, the range noted here reflects the variability inher- ent in the two assay methods.

To understand the reproducibility of the RT-PCR assay further, additional replicates of some of the

Todd et al.: Quantitation of HIV plasma by branched DNA and RT-PCR assay methods 237

specimens described in Table 2 were included in assay runs 1 and 2. The results (in 000s) were: (run 1) A, 143.6; B, 78.4; C, 62.8: D, 15.8; E, 14.1: F, 5.7; G, 4.0; H, 1.6; I, 1.1: (run 2) F. 4.3: G, 2.1; H, 1.1; I, 0. These data taken together with the RT-PCR results in Table 2 were used to generate estimates of random error variance for RT-PCR (overall inter-assay), above and below the detection limit of the bDNA assay. To achieve this, the samples were separated into two groups, those detected by both assays (group 1) and those detected only by RT-PCR (group 2). The variance estimates were: group 1, 0.33076 and group 2, 0.28463376. These values were used to predict the 95% tolerance limit for the difference between two log quantitations and were: group 1; 8.1 and group 2; 6.9. These results indicate that for the Roche Biomedical Laboratories RT-PCR procedure, a 6.9-8.1 difference in quantitation between two specimens would be required to be statistically significant.

2.5 /

0.5 j

--~. -ii--T--,

4 4.5 Log5 5.5 6 6.5 7

NW RNA Co@edmi

Figure 1. Quantitation of RNA transcripts representing HIV subtypes A-E using the bDNA assay. ---O---, Subtype A; -0-, subtype B; ---m---, subtype C; -•O-, subtype D; -W-, subtype E.

Effect of genotype on quantitation

The effect of genotypic variation on RNA quantitation was assessed in two separate sets of experiments. In the first set, a dilution panel consisting of RNA transcripts (diluted into proteinase k treated plasma), represent- ing pol gene sequences from subtypes A-E, were tested with the bDNA assay. These RNA transcript preparations were independently quantitated using three separate analytical procedures to establish the actual quantitation. The mean quantitations obtained over six bDNA assay runs, for each subtype dilution series, were obtained and the results of a representa- tive assay run are presented in Figure 1. The largest variation in quantitation between the subtypes was 1.5; however, there was not a significant (PcO.05) subtype effect on quantitation (P = 0.33; one-way ANOVA). This finding demonstrated that the bDNA assay was able to quantitate equally HIV RNA present in the major virus subtypes at concentrations that span the dynamic reportable range for the assay. The second set of experimentation focused on a direct comparison between bDNA and RT-PCR. RT-PCR amplification is based on primers located in the gag gene” and since the RNA transcript preparations represented pol gene sequences, the transcripts could not be used. Instead. stock HIV culture isolates, representing subtypes A, C-F, were diluted into HIV negative plasma and tested with both assays. The dilutions, which were based on bDNA assay quantitation of the culture stocks, were targeted to yield 40 X 103 RNA Eq ml-‘. Two distinct isolates for each subtype (see Table 3) were tested on two separate occasions that were greater than 2 months apart. The results of this comparison are presented in Table 3, and show that with the bDNA assay, quanti- tation ranged from 28 X 10’ to 56 X 101 Eq ml-‘, whereas RT-PCR mediated quantitation ranged from 240 to 217 X lo3 copies ml-‘. The largest ranges in quantitation across the subtypes were 2-fold for bDNA and 904-fold for RT-PCR. These differences in quanti- tation of the HIV subtypes were confirmed when identical specimens were tested by bDNA and RT- PCR on a second occasion.

Table 3. The effect of HIV subtype on RNA quantitation by bDNA and RT-PCR

Subtype* Isolate Homologyt bDNA RT-PCR RNA % RNA eq ml-’ (X 103) copies mt-’ (X 1031

A A C C D D E E F F

DJ263 89 28 0.7 DJ258 so 30 0.2

ZAM20 88 52 217 ZAM18 89 31 84 UG270 92 43 96 UG274 92 53 104 CM235 89 38 18 CM241 89 56 45

162.3069 ND 43 41 163.3070 ND 33 13

“Specimens consisted of cultured virus diluted into HIV negative plasma. tpercentage nucleotide homology to the pol gene of subtype 6 (strain SFZ) ND, Determination not made.

238 Serodiagn. Immunother. Infect. Disease 1994; 6: No 4

Discussion

The potential clinical applications of quantitative HIV RNA determinations are centred around patient management and use as a surrogate marker for clinical endpoints in clinical trials of new therapeutic agents 1,*+,5.20.*4~?S.*7. In both cases, the utility of the measurement resides in being able to discern changes in viral burden in plasma specimens taken over a defined time period. Reproducibility of the assay method used to make the quantitative measurements directly affects the magnitude of a change that can be measured and be statistically significant. In the present study we have evaluated the reproducibility of the bDNA assay with 96 assay runs over 6 months using three different lots of kits. Depending on the kit lot, the overall reproducibility analysis indicated that a 1.65-1.81-fold change in RNA levels would be statisti- cally significant. This form of real-time analysis repre- sents the application in a clinical setting involved in long term patient monitoring. In contrast, RNA measurements for clinical trials are more likely to be performed in a batch mode. Under this latter situation, the bDNA assay can discern a 1.24-l .34-fold change as statistically significant. Taken together, these data show that the bDNA assay can measure less than 2-fold changes in RNA levels. Holodniy et a1.“4, using RT- PCR, have recently demonstrated that HIV virion associated plasma RNA levels display small diurnally and weekly fluctuations. Due to the inherent variabil- ity of RT-PCR, the magnitude of the natural fluctua- tions was not clear and more work is required to determine if 2-3-fold changes in RNA levels, which can be measured with the bDNA assay, have clinical relevance. However, it should be noted that small changes in plasma HIV RNA levels have been noted during immunomodulatory therapy*” and that these changes were closely associated with initiation of therapy. Small decreases in HIV RNA levels are likely to be associated with a partial response to antiviral therapy. In contrast, small increases in HIV RNA levels could be associated with an early prediction of antivi- ral failure, wherein, resistance leads to increases in viral burden’. In contrast, the RT-PCR assay performed at Roche Biomedical Laboratories demonstrated a larger variability, such that the limit for being able to discern changes in RNA levels statistically was 6.9-8.1-fold, depending on the level of RNA in the specimen.

Accuracy of an assay is important when it is desired to know the ‘true’ quantity, not simply the relative concentration. For nucleic acid quantitative analyses to be accurate, the effect of sequence variation must be assessed. Although testing of geographically distinct specimens is a first step, for HIV, where clonal spread has occurred in several areas, insufficient sampling could lead to erroneous results. To address this issue, we chose to test the most diverse sequences by using RNA transcripts and clinical isolates of known subtype variants. Using RNA transcripts prepared from mole- cularly cloned sequences of the pal gene from HIV

subtypes A-E, the bDNA assay accurately quantitated the subtypes. Since the subtypes represent maximum genotypic variation, one can expect that genotypic differences within a specific subtype should not affect bDNA assay quantitation. Although for the field isolates, the percentage homology in pof gene sequences ranged from 88-92%, compared to that of subtype B (strain SF2). the quantitation values showed large variability when measured with the RT-PCR assay. For example, the mean values for subtypes A and C were 460 copies ml-’ and 151 X 10’ copies ml-’ respectively, resulting in a 328-fold range and a 904- fold range between isolates DJ258 (subtype A) and ZAM20 (subtype C). In contrast, the range in quanti- tation for the same specimens using the bDNA assay was 1.43-fold. These findings are consistent with the design of the probes used in the bDNA assay, wherein 49 different probe sequences are used and sequence degeneracy was employed to minimize subtype effects”. The disparity in quantitation seen between the subtypes with RT-PCR is clear. This observation should be confirmed using gag transcripts prepared from cloned gag gene sequences containing RT-PCR primer binding sites,

The RT-PCR assay displayed a sensitivity greater than the bDNA assay: however, this determination was based on the dilution of a single specimen. Since quantitation was seen to vary greatly between subtypes, it is reasonable for sensitivity to vary likewise. In fact, the bDNA assay should be 5-10 times more sensitive than the RT-PCR assay for HIV subtype A. Finally, the increased sensitivity was obtained with a loss in assay reproducibility at the lower limits of detection. For example, five quantitative determinations for dilution panel member I resulted in a mean value of 628 copies ml-‘; however, the standard deviation was 550 copies ml-’ and the individual determinations ranged from 0 to 1.3 X lo3 copies ml-‘.

This study has demonstrated that quantitative HIV RNA assays based on RT-PCR and bDNA technolo- gies have different attributes and performance charac- teristics that need to be taken into account. The RT-PCR assay appears to quantitate some samples of HIV at levels undetected by the bDNA assay: however, the sensitivity is gained with a loss in repro- ducibility. The bDNA assay can measure much smaller changes in RNA levels than the RT-PCR assay. Finally, unlike the bDNA assay, the accuracy and sensitivity of RT-PCR appears to be influenced greatly by genotypic variation. Consequently, when quanti- tated by RT-PCR assays, the level of sequence homo- logy or genotype of the infecting virus may need to be known before conclusions can be made about RNA levels and disease progression and therapeutic inter- vention.

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