jcm accepts, published online ahead of print on 6 may 2009 j. … · 2009. 5. 6. · jcm accepts,...

1

Title: 1

An evaluation of the Abbott m2000 RealTime HIV-1 assay for HIV viral load 2

monitoring in South Africa compared to existing technologies: Roche COBAS 3

Ampliprep/COBAS Amplicor; Roche COBAS Ampliprep/COBAS TaqMan HIV-1, 4

and BioMerieux NucliSENS EasyQ HIV-1. 5

6

Running title: Evaluating the Abbott m2000 RealTime HIV-1 assay 7

8

Authors: 9

Lesley E Scott* 10

Department of Molecular Medicine and Haematology, University of Witwatersrand, 11

Faculty of Health Sciences, School of Pathology, 7 York road Parktown, Room 3B20, 12

Johannesburg 2000, South Africa. +27 11 489 8567 (tel), +27 484 5812 (fax), email: 13

[email protected] 14

Lara D Noble 15


Faculty of Health Sciences, School of Pathology and National Health Laboratory 17

Services, 7 York road Parktown, Room 3B20, Johannesburg 2000, South Africa. 18

Jackie Moloi, 19




23

Copyright © 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.J. Clin. Microbiol. doi:10.1128/JCM.01761-08 JCM Accepts, published online ahead of print on 6 May 2009

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Linda Erasmus 1




Willem D.F Venter 5

Reproductive Health and HIV Research Unit, University of Witwatersrand, 6

Johannesburg, South Africa 7

Wendy Stevens 8




12

Abstract 13

Implementation of antiretroviral therapy demands the need for increased access to viral 14

load (VL) monitoring. Newer real-time VL testing technologies are faster with larger 15

dynamic ranges and fully automated extraction to benefit higher throughputs in the 16

resource poor environments. The Abbott RealTime HIV-1 assay was evaluated as a new 17

option for HIV-1 subtype C testing in South Africa and compared to existing assays 18

(CAP/CTM HIV-1, CAP/CA HIV-1, NucliSENS HIV-1) within a high throughput 19

laboratory. The RealTime HIV-1 total assay precision is acceptable at all viral load 20

ranges. This assay compares most favourably with the CAP/CTM HIV-1 (R2=0.904), 21

with a low SD of difference (0.323copies/ml). The bias against comparator assays ranges 22

from -0.001copies/ml to -0.228copies/ml. Variability in reporting VL on a twenty 23

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member subtype panel compared to other assays was noted with subtypes G and CRF02 1

AG. The RealTime HIV-1 assay performs 93 samples per day with minimal manual 2

preparation, less staff and minimisation of contamination through automation. This assay 3

is suitable for subtype-C VL quantification in South Africa. 4

Word count: 168 5

6

Key Words: HIV-1 viral load quantitation, Real Time PCR, high throughput Subtype-C 7

8

Introduction 9

South Africa bears a huge burden of HIV with an estimated 5.5 million infected 10

individuals (9). In September 2007 approximately 329 000 individuals received 11

antiretroviral (ARV) treatment on the National Program (17, 18). Globally viral load 12

(VL) testing has been used to monitor treatment, determine prognosis and risk of disease 13

progression and to identify treatment failure (48). South African treatment guidelines use 14

viral load results to determine time of switch from first to second line drug regimen (16). 15

Work from the region has demonstrated that the use of VL monitoring together with 16

targeted adherence approaches result in significant conservation of the first line drug 17

regimen (2). In addition it has been demonstrated that switching therapy based on clinical 18

and immunological criteria alone results in the development of more complex resistance 19

profiles (44) thus reinforcing the value of viral load monitoring. Initial WHO guidelines 20

for ARV treatment in resource poor settings referred to the use of VL as optional (19). 21

There has been a recent shift in thinking with the revised guidelines referring to VL 22

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testing as desirable in resource limited settings (11), with greater international effort 1

being focussed on improving access to virological testing. 2

Nucleic acid amplification technologies are used in most settings for VL testing, and are 3

either based on signal (30) or target amplification methodological approaches (24). The 4

trend now is towards using real-time detection of amplicons (45), which simply means 5

that detection is done as the product accumulates during the exponential phase of the 6

reaction. Newer real-time technology options are faster, have higher throughputs, larger 7

dynamic ranges and fully automated extraction steps (45). To ensure that the South 8

African National Health Laboratory Service (NHLS) can cope with the high volumes of 9

anticipated samples as the treatment program matures, several automated, real-time 10

monitoring assays have been explored in-country. 11

In this study we investigated and compared the performance of the Abbott RealTime 12

HIV-1 assay (abbreviated to RealTime HIV-1) on the m2000sp/m2000rt automated 13

extraction platform (Abbott Molecular Inc., Des Plaines, Illinois, USA) to the COBAS 14

Ampliprep/COBAS TaqMan HIV-1 version 1 (abbreviated to CAP/CTM HIV-1) (F. 15

Hoffmann-La Roche, Diagnostics Division, Grenzacherstrasse, Basel,Switzerland), the 16

NucliSENS EasyQ / EasyMag HIV-1 (abbreviated to NucliSENS HIV-1) version 1.1 17

(bioMérieux, Boxtel, Netherlands) and the Ampliprep/COBAS Monitor standard HIV-1 18

assay (abbreviated to CAP/CA HIV-1) assays which are already established within the 19

South African laboratory setting (36, 37). 20

The first three assays mentioned are real-time platforms with fluorescence detection 21

systems, and the CAP/CA HIV-1 is based on endpoint detection. Previous publications 22

report on the performance of the CAP/CTM HIV-1 (12, 21, 28, 29, 31) and the 23

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NucliSENS HIV-1 (8, 13, 36, 37) assays . The Abbott RealTime HIV-1 assay has been 1

validated previously against the Roche HIV-1 Monitor assay (using a manual RNA 2

extraction step) and the CAP/CTM HIV-1 assays using the m1000 system, which is less 3

automated than the m2000sp system used in this study (6, 15). Other studies have 4

compared the Abbott RealTime assay to the Roche COBAS Monitor assay for HIV-1 and 5

HCV (32, 41, 42). Swanson and colleagues (2006) compared the fully automated m2000 6

system RealTime HIV-1, the CAP/CA 1.5, the Versant HIV-1 RNA 3.0 and the Abbott 7

LCx HIV-1 RNA Quantitative systems (42). A recent study has also compared the 8

RealTime HIV-1 to the NucliSENS HIV-1 for HIV-1 clades prevalent in China (47). 9

Although these reports published the performance of the Abbott viral load assay alone or 10

in comparison to several existing VL assays and on different subtypes, the study 11

presented here is the first evaluation of the RealTime HIV-1, the NucliSENS HIV-1, the 12

CAP/CTM HIV-1 and the CAP/CA HIV-1 on predominantly HIV-1 subtype C in South 13

Africa. 14

15

Materials and Methods 16

Sample Collection and Storage 17

Whole blood K3EDTA samples were collected from 150 consenting patients presenting 18

for a routine HIV viral load test at the ARV clinic of the Johannesburg Hospital, South 19

Africa. The plasma was extracted within 4 hours of collection and stored in aliquots at 20

-80°C until further processing. They were then thawed, centrifuged at 3000g for 5 21

minutes and specified volumes added to each assay as per manufacturer’s instructions. 22

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This study was conducted as per ethics approval (M061105) from the Human Ethics 1

Committee of the University of the Witwatersrand (Johannesburg, South Africa). 2

3

Viral load methodologies 4

All assays were performed by dedicated, trained operators in good laboratory practice 5

(GLP) compliant laboratories according to the manufacturer’s instructions. The m2000 6

system is based on real-time polymerase chain reaction (PCR) technology and consists of 7

two instruments: the Abbott m2000sp (automated extraction) and the m2000rt (real-time 8

PCR) instrument that amplifies and detects HIV-1 amplicons. The system automates a 9

variety of manual processing steps, such as pipetting, helping to reduce the hands-on time 10

required to prepare patient samples. The detection of the PCR product in real-time uses a 11

relatively new probe design: partially double-stranded probes. The probe consists of two 12

DNA fragments of differing lengths: the longer fragment is complementary to the target 13

DNA and is bound to a fluorescent marker, while the shorter fragment holds the quencher 14

molecule. When the target DNA is not present, the long probe binds to the quencher 15

probe and no fluorescence is detected; when the target DNA is present, the long probe 16

binds preferentially to the target DNA and is able to fluoresce to give a quantifiable 17

signal. The advantage of these probes is their increased tolerance to mismatches (20, 23), 18

which is particularly useful in viruses, such as HIV, which are highly mutable. Table 1 19

summarises salient characteristics of each assay. 20

Statistical analysis 21

To accommodate the differences in the reportable ranges between the endpoint and real-22

time assays as outlined in Table 1, and prevent skewing of data, all the values were 23

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modified according to the CAP/CA HIV-1 assay limits. Any continuous value 1

<400copies/ml was changed to read 400copies/ml and any value >750 000copies/ml was 2

changed to read 750 000copies/ml. (6). These adjusted values were then log10 3

transformed. Data was sorted according to qualitative and quantitative values. Samples 4

not detected by one or more assays were excluded from the continuous data analysis, but 5

were used for the qualitative analysis. All assays were reported in copy number/ml. 6

Precision and accuracy of the RealTime HIV-1 assay 7

The intra-variability and inter-variability for only the RealTime HIV-1 assay was 8

confirmed in this study on local specimens and compared to the manufacturer’s claims. 9

Three samples were diluted in HIV negative human plasma (to concentrations of 100,000 10

copies/ml, 1000 copies/ml and 100 copies/ml) and stored in twenty five 1ml aliquots. 11

Intra-variability was measured from five aliquots tested in one run. Inter-variability was 12

measured by analysing a different run each day for 5 days (5 runs). Variability was 13

assessed using the standard deviation (SD) and coefficient of variation (CV) on both the 14

absolute and Log transformed data, which are reported as a range (lowest to highest) for 15

the 5 runs. These values were compared to the manufacturer’s claims. As a guide, a 16

SD≤0.15 on the Log transformed values and a CV≤35% on the absolute values is 17

generally acceptable (5). 18

Accuracy was determined for continuous values using the Bland-Altman (3) (difference 19

plot) and the percentage similarity (33) that measure amount of agreement between 20

assays. Linear regression is also reported with the equation of the line, R2 and p-value for 21

the intercept. The Bland-Altman records the bias (with the limits of agreement) between 22

two methods. The distance between the two limits of agreement is also reported, which is 23

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the value of both limits of agreement added (ignoring their signs) and divided by 2, and 1

shows the overall difference for 95% of the data. The bias of all the assays converted to 2

IU/ml with confidence intervals is also reported. The percentage outliers >log0.5 and 3

>log1.0 were also calculated for each comparison. The percentage similarity (33) 4

determines the percentage accuracy between two or more methods using the mean 5

percentage similarity and the precision between two methods using the percentage 6

similarity SD. Both these values are reported as overall agreement between methods as a 7

percentage similarity CV. 8

Qualitative analysis 9

The assays were also evaluated with respect to the following qualitative parameters: 10

sample handling, time to reportable result, capacity (number of patient samples and 11

controls per run) (extraction and detection), robustness of tests. All analyses were 12

performed using Microsoft™ Excel and SAS version 9.1, Enterprise guide 3.1. 13

14

Subtype panel 15

A 20-member subtype panel derived from HIV-1 infected blood donors in Cameroon, 16

South Africa, Thailand and Uganda was obtained from the Abbott Global Surveillance 17

Program (Abbott Diagnostics, Abbott Park, IL) and consisted of five CRF02-AG, three 18

CRF01-AE, three subtype G, three subtype A, three subtype D and three subtype F . 19

Subtype assignments were based on sequence analysis of gag p24, pol IN, and env gp41 20

IDR as previously described (40), thus including the target regions of all the assays being 21

evaluated. The panel was designed to represent common group M, non-B subtypes and 22

circulating recombinant forms. The panel was stored at –80oC prior to testing and was 23

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only performed on the three real time assays and not the CA platform. The panel had not 1

previously been tested using the CAP/CTM HIV-1 or NucliSENS HIV-1 assays. 2

3

Results 4

RealTime HIV-1 precision analysis 5

The intra-variability for the RealTime HIV-1 was: SD=0.04-0.07, CV=0.91-1.4%, 6

absolute CV=11.3-17.9% for Log5 copies/ml; SD=0.05-0.09, CV=1.6-2.9%, absolute 7

CV=11.7-18.9% for Log3 copies/ml; and SD=0.04-0.16, CV=1.7-7.9%, absolute 8

CV=9.4-34.0% for Log2 copies/ml. The inter-variability was: SD=0.07-0.11, CV=1.5-9

2.2%, absolute CV=18.6-28.8% for Log5 copies/ml; SD=0.09-0.15, CV=2.5-4.7%, 10

absolute CV=18.7-34.7% for Log3 copies/ml; and SD=0.13-0.25, CV=7.7-10.7%, 11

absolute CV=32.5-52.1% for Log2 copies/ml. The intra assay variability appears stable 12

throughout the runs for the entire range of log values, although as expected the assay 13

appears less precise in the lower viral load range (log2.0), but with only one run 14

producing a variability CV >35% on the absolute result. This increased variability 15

appeared in run 5 contributing to increased inter-variability, and may reflect a loss of 16

viral load on storage, which becomes more noticeable at the lower limit. Total assay 17

variability, according to the Abbott manufacturer claim (<0.3 SD copies/ml) however, is 18

acceptable at all viral load ranges. 19

20

21

22

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RealTime HIV-1 accuracy analysis 1

Description of the data as continuous and discrete variables 2

The 150 samples yielded 67 continuous values and 83 sample results that registered 3

<LDL (lower than detectable limit) or TND (target not detected). The latter reflects 4

patients attending the clinic for monitoring viral loads on ARV treatment. These discrete 5

results yielded n=10 continuous values (12%) for NucliSENS HIV-1 (2 values > 6

reported linear limits; 120IU/ml, 750IU/ml) , n=5 continuous values (6%) for CAP/CTM 7

HIV-1 (51copies/ml, 53 copies/ml, 68 copies/ml, 146 copies/ml, 155 copies/ml) , n=3 8

continuous values (3.6%) for the RealTime HIV-1 (45 copies/ml, 50 copies/ml, 167 9

copies/ml) and n=0 continuous values (0%) for CAP/CA HIV-1. 10

The remaining continuous values were capped according to the CAP/CA assays’ limits 11

(400-750 000copies/ml). The mean and median values for the respective assays were: 12

log4.72copies/ml (NucliSENS HIV-1), log 4.95copies/ml (CAP/CTM HIV-1), log 4.72 13

copies/ml (RealTime HIV-1) and log 4.94 (CAP/CA HIV-1). The medians for the four 14

assays ranges from 4.94-5.44copies/ml showing the majority of samples in this data set 15

were in the upper range. 16

17

The comparative statistics are detailed in Table 2 (a, b, c, d), where each assay is 18

compared to all the other assays. The Bland-Altman difference plots for these assay 19

combinations is also represented in Figure 1 (a,b,c,d). The RealTime HIV-1 assay 20

compared to all assays shows the ‘no-intercept’ model can be applied when this assay is 21

compared to the NucliSENS HIV-1. Comparison to the CAP/CTM HIV-1 and CAP/CA 22

HIV-1 have a significant intercept (p<0.05). However the CAP/CTM HIV-1 coefficient 23

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of determination is the highest showing the equation of the line between these two assays 1

represents 90.8% of the data. The smallest bias in copies/ml is with the NucliSENS HIV-2

1 and the smallest bias in IU/ml is with the CAP/CTM HIV-1. This latter comparison also 3

has the smallest variability (lowest SD) and also the smallest distance over the 95% limits 4

of agreement. The lowest number of outliers <log0.5copies/ml is the comparison with 5

CAP/CTM HIV-1, but with CAP/CA HIV-1 shows no clinically relevant outliers 6

(>log1.0 c/ml). The RealTime HIV-1 and CAP/CTM HIV-1 assay comparison has the 7

lowest percentage similarity CV (overall accuracy and precision). 8

The comparison between the NucliSENS HIV-1 and all assays shows the ‘no-intercept’ 9

model can be applied to the comparison with RealTime HIV-1, and the highest R2 occurs 10

with the CAP/CA HIV-1. The smallest bias is with the RealTime HIV-1 reporting in 11

copies/ml, but with CAP/CA HIV-1 reporting in IU/ml. The CAP/CA HIV-1 also has the 12

lowest variability (SD) and the smallest distance over the limits of agreement. The fewest 13

outliers <log0.5c/ml is with the RealTime HIV-1 and the CAP/CA HIV-1, with no 14

clinically relevant outliers (>log1.0c/ml) compared to the CAP/CTM HIV-1. The 15

NucliSENS HIV-1 and the RealTime HIV-1 assay comparison has the lowest percentage 16

similarity CV (overall accuracy and precision). 17

The comparison between CAP/CTM HIV-1 and all other assays shows the ‘no-intercept’ 18

model can be applied between all three other assays (p>0.05), with the highest coefficient 19

of determination with the RealTime HIV-1 assay. The smallest bias is between 20

NucliSENS HIV-1 and RealTime HIV-1 for copies/ml and NucliSENS HIV-1 for IU/ml. 21

The least variability is with the RealTime HIV-1and the smallest differences for 95% of 22

the data pairs with RealTime HIV-1 as well the fewest outliers <log0.5c/ml, but no 23

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clinically relevant difference (>log1.0c/ml) with the NucliSENS HIV-1 assay. The 1

CAP/CTM HIV-1 and the RealTime HIV-1 assay comparison has the lowest percentage 2

similarity CV (overall accuracy and precision). 3

The comparison between CAP/CA HIV-1 and all other assays shows the ‘no-intercept’ 4

model can be applied to all assays, with the highest coefficient of determination with 5

CAP/CTM HIV-1. The smallest bias is with the NucliSENS HIV-1 and the RealTime 6

HIV-1 for copies/ml and with NucliSENS HIV-1 for IU/ml. The lowest variability is with 7

the CAP/CTM HIV-1, which also has the smallest distance over the limits of agreement. 8

The fewest outliers <log0.5c/ml is with NucliSENS HIV-1 and no clinically relevant 9

outliers (>log1.0c/ml) with the RealTime HIV-1. The CAP/CA HIV-1 and the CAP/CTM 10

HIV-1 assay comparison has the lowest percentage similarity CV (overall accuracy and 11

precision). 12

Overall the assay combinations with the highest coefficient of determination are the 13

RealTime HIV-1 and the CAP/CTM HIV-1 (R2=0.908). The smallest bias is between the 14

RealTime HIV-1 and NucliSENS HIV-1 (-0.001 copies/ml) and between NucliSENS 15

HIV-1 and CAP/CA HIV-1 (0.067IU/ml). The least variability in differences is between 16

RealTime HIV-1 and the CAP/CTM HIV-1 (SD-0.323copies/ml), which also has the 17

smallest distance between limits of agreement (0.646copies/ml). The fewest total number 18

of outliers (both >log0.5copies/ml and >log1.0copies/ml) occurs between RealTime HIV-19

1 and CAP/CTM HIV-1 assays, whereas the comparisons between CAP/CTM HIV-1 and 20

NucliSENS HIV-1 and between RealTime HIV-1 and CAP/CA HIV-1showed no 21

clinically relevant outliers (>log1.0c/ml). In terms of overall accuracy and precision 22

(measured by the percentage similarity), the assay combinations with the lowest 23

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percentage similarity CV were between the CAP/CTM HIV-1 and CAP/CA HIV-1 1

(3.7%) and between the RealTime HIV-1 and the CAP/CTM HIV-1 (3.7%). 2

3

Subtype Panel analysis 4

All three real-time assays quantified the 20 members of the subtype panel. The 5

NucliSENS HIV-1 reported one subtype F specimen as invalid, but on repeat testing 6

yielded 3,300 IU/ml. The NucliSENS HIV-1 values generated lower values for many of 7

the specimens and the RealTime HIV-1 tended to generate higher values than the other 8

assays. Only four samples (two subtype G and two subtype CRF02-AG) produced 9

>log1.0 differences between assay. Three were between the RealTime HIV-1 and the 10

NucliSENS HIV-1and one between the RealTime HIV-1 and the CAP/CTM HIV-1. 11

12

Qualitative assay analysis 13

Robustness of Assays 14

All assays gave valid results on the patient specimens, with one invalid on the CAP/CTM 15

HIV-1 assay (repeat yielded TND similar to other assays). Table 3 outlines the 16

throughput of samples for all four assays that can be performed in the Johannesburg 17

laboratory together with the number of staff required for each method. Throughput is 18

highest for the NucliSENS HIV-1 followed by the RealTime HIV-1 assay. Although the 19

TaqMan48 was used in this study, a TaqMan 96 is available on the market, which would 20

generate 84 patient results per run (including 12 controls per batch of 96). Staff 21

requirements are greater for the NucliSENS HIV-1 assay. The RealTime HIV-1 and the 22

CAP/CTM HIV-1 are also assays that can be performed in a laboratory area without 23

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separating the extraction and amplification components. Both the NucliSENS HIV-1 and 1

the RealTime HIV-1 instruments have small footprints compared to the Roche 2

instruments, and an added advantage to the RealTime HIV-1 assay is the utilization of the 3

m2000rt for additional real-time assay expansion. 4

5

Discussion 6

The South African ARV program is unique in a resource limited setting for the following 7

reasons: viral load testing is used currently to switch a failing drug regimen, the result is 8

required within 72 hours of collection, the volumes of VL samples that need to be 9

processed is enormous and the epidemic involves predominantly a single subtype, HIV-1 10

subtype C. Routine viral load testing laboratories nationally, as in many resource 11

constrained countries are characterised by a shortage of skilled technical staff and limited 12

laboratory space. The requirements of the VL testing program are thus: i) an accurate, 13

reliable, robust assay, ii) a high throughput platform to facilitate rapid turnaround time, 14

iii) small instrument footprints, iv) ability to use less skilled staff, v) simpler transport 15

requirements and vi) affordable cost per reportable result. Many VL evaluations have 16

been conducted locally (10, 25, 34, 36-39), but this represents the first study investigating 17

the newly available Abbott RealTime HIV-1 automated (m2000sp and m2000rt) assay, in 18

a high throughout laboratory in the South African environment of HIV-1 subtype C, in a 19

side by side comparison with three existing platforms. 20

The RealTime HIV-1 assay variability appears acceptable across the viral load ranges 21

investigated, which concurs with other studies (13, 27, 32). There is increased variability 22

in the lower viral load range (log 2), which is typical of most viral load assays (5). The 23

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RealTime HIV-1 showed least fluctuation (n=3, 3.6%) at the lower limit of detection 1

compared to the other real-time assays. The statistical models concur that the Abbott 2

RealTime HIV-1 compares favourably with all the existing assays tested in this 3

laboratory, and performs overall best with the CAP/CTM HIV-1. Similar comparisons 4

between these two assays have been reported in the literature (6, 46). The comparison 5

overall between the RealTime HIV-1 and the NucliSENS HIV-1 was least favourable, 6

which has been similarly noted in a Chinese study (47). Our study also highlights the 7

changes that can occur in the bias between assays when converting between copy number 8

and IU and that caution is required when changing between assays or reporting units. One 9

such difference is noted between the CAP/CTM HIV-1 and the CAP/CA HIV-1, where 10

the bias changed from -0.35copies/ml to 0.16IU/ml. 11

12

The genetic diversity of HIV-1 presents a significant challenge to development of assays 13

capable of reliably quantifying all strains of the virus, (7, 15, 38). In the present study, 14

we utilized a 20-member panel comprising subtype A, D, F, G, CRF01_AE and 15

CRF02_AG strains over and above the local subtype C samples to evaluate the 16

comparative performance of the three real-time assays. All three real-time assays were 17

able to quantify all panel members. All three systems adequately detected subtypes B, D 18

and F. The subtypes >log1.0 variance were subtype G (n=2) and CRF02_AG (n=2) with 19

the NucliSENS HIV-1 and RealTime HIV-1 and CRF02_AG (n=1) with the CAP/CTM 20

HIV-1 and RealTime HIV-1. These differences for the NucliSENS HIV-1 and CAP/CTM 21

HIV-1 have been reported previously (7, 8, 13-15, 35). 22

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The qualitative characteristics of the RealTime HIV-1 assay compares favourably with 1

existing assays in that it performs 93 samples in one shift (reportable results in one 8 hr 2

day). Although this is less than the current NucliSENS technology, the RealTime HIV-1 3

requires less manual preparation (42) and therefore less staff. In addition the 4

amplification procedure can be left overnight which increases the number of tests in a 5

cycle although not in a single shift. In this study the RealTime HIV-1 methodology was 6

found easy to perform with no sample repeats. Controls are also supplied with each batch 7

and the number of controls is also the same for 48 or 96 tests performed. This assay 8

showed overall good performance and potential for use in populations with a 9

predominance of HIV-1 subtype C. It is reliable with minimisation of contamination 10

through automation, rapid turn-around time (with little manual time requirements), and 11

instrument utilization for assay expansion (hepatitis C (46)), Chlamydia trachomatis, 12

Neisseria gonorrhoeae (26). 13

14

Acknowledgements: 15

Abbott Molecular, Roche Diagnostics and BioMérieux for assay reagents and 16

consumables and critical comments on the pre-published manuscript. Dr John Hackett, Jr 17

from Abbott Diagnostics, Abbott Park, IL, USAfor assistance with the subtype panel and 18

manuscript preparation. Margaret Langeveldt for collecting the EDTA specimens from 19

consenting adults. The National Health Laboratory Service PCR Laboratory staff in the 20

Department of Molecular Medicine and Haematology for performing the routine analysis 21

on the CAP/CTM HIV-1, CAP/CA HIV-1 and NucliSENS HIV-1 assays. 22

23

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Support for this study: 1

This publication was made possible by the generous support of the American people 2

through the US Agency for International Development. The contents are the 3

responsibility of the authors and do not necessarily reflect the views of USAID or the US 4

government. 5

6

7

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Table 1: Characteristics of Abbott RealTime HIV-1, NucliSENS HIV-1, CAP/CTM HIV-1

1 and the CAP/CA HIV-1 Assays. 2

Abbreviation RealTime HIV-1 NucliSENS HIV-1 CAP/CTM HIV-1 CAP/CA HIV-1

Full Name

Abbott RealTime

HIV-1

NucliSENS

EasyMag/ EasyQ HIV-1

COBAS Ampliprep/

COBAS TaqMan HIV-1

COBAS Ampliprep/

COBAS Amplicor

HIV-1 Monitor 1.5

(Standard)

Target Pol/IN HIV-1 Gag HIV-1 Gag HIV-1 Gag HIV-1

Extraction

Automated m2000sp

RNA extraction.

Automated EasyMag

total nucleic acid

extraction

Automated COBAS

Ampliprep RNA

extraction

Automated COBAS

Ampliprep RNA

extraction

Amplification &

Detection

Real-time PCR: Non

competitive

fluorescent detection. .

(20, 23),

Real-time NASBA PCR

with fluorescent

detection (molecular

beacons with reporter

and quencher molecule

(4, 43)

Real-time PCR:

fluorescent detection

using TaqMan probes

(5’-nuclease probe

breakage) with

fluorophore and

quencher(22).

Manual loading. PCR

with Endpoint

detection: photometric

quantification of an

enzyme-linked colour

reaction (1)

Amplification

and Detection

m2000rt NucliSENS Analyser Cobas Taqman Cobas Amplicor

Actual plasma

volume used for

extraction

0.8ml 1.0ml 1.0ml 0.35ml

Assay sample

input volume

0.6ml 1.0ml 0.85ml 0.20ml

Reported result

Copies or IU/ml:

detectable

IU/ml or copies/ml:

Detectable within linear

Copies or IU/ml/ml:

detectable quantifiable,

Copies or IU/ml

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quantifiable,

detectable beyond

upper quantifiable

limit (>UQL/

<40copies/ml),

undetectable

range,

Not detectable or below

detectable limit (<LDL)

detectable beyond upper

quantifiable limit

(>UQL <40copies/ml),

undetectable

Linear Range 40 – 1x107copies/ml 100 – 3x10

6 IU/ml 40-1x10

7 copies/ml 400-750,000 copies/ml

Conversion 1 copy = 1.74IU 1copy = 1 IU* 1copy = 1.7IU 1 copy = 1.96IU

95% detection 39copies/ml (68IU/ml) 250IU/ml 40copies/ml (68IU/ml) 400copies/ml

# PCR: polymerase chain reaction 1

*Confirmed by supplier’s memorandum 2

3

4

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Table 2: Bland-Altman and percentage similarity statistical parameters for measuring 1

agreement between all assays. All assays are reported in copies/ml unless otherwise 2

stated for n=67. 3

(a)RealTime HIV-1 versus the NucliSENS HIV-1, CAP/CTM HIV-1 and CAP/CA 4

assays. 5

6

RealTime HIV-1 vs NucliSENS HIV-1 CAP/CTM HIV-1 CAP/CA HIV-1

Linear Regression

Equation y=0.939x+0.286 y=0.899x+0.348 y=0.942x+0.5

P-value on intercept p=0.195 p=0.046 p=0.017

R2 0.868 0.908 0.883

Bland-Altman

Bias in copies/ml -0.001(-0.098;0.094) 0.125(0.045;0.20) -0.228(-0.318;-0.137)

Bias in IU/ml 0.233(0.136;0.329) 0.136(0.057;0.125) 0.30(0.209;0.391))

SD 0.395 0.323 0.371

Limits of Agreement (0.789;-0.791) (0.771;-0.521) (0.514;-0.97)

Distance over limits 0.79 0.646 0.742

% outliers >log0.5c/ml 14(20.9%) 7(10.4%) 22(32.8%)

% outliers >log1.0c/ml 2(2.98%) 2(2.98%) 0

Percentage similarity

Mean (%) similarity 100.2% 99% 102.8%

SD (%) similarity 4.73% 3.66% 4.7%

% CV similarity 4.72% 3.7% 4.57%

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(b) NucliSENS HIV-1 versus CAP/CTM HIV-1, RealTime HIV-1 and CAP/CA 1

NucliSENS HIV-1vs CAP/CTM HIV-1 RealTime HIV-1 CAP/CA HIV-1

Linear Regression

Equation y=0.867x+0.5 y=0.92x+0.356 y=0.936x+0.526

P-value on intercept p=0.021 p=0.104 p=0.011

R2 0.857 0.868 0.886

Bland-Altman

Bias in copies/ml 0.126(0.027;0.225) 0.002(-0.095;0.098) -0.226(-0.316;-0.137)

Bias in IU/ml -0.096(-0.195;0.002) -0.23(-0.329;-0.137) 0.067(-0.022;0.157)

SD 0.406 0.395 0.366


Distance over limits

0.812 0.79 0.732

% outliers >log0.5c/ml 18(26.9%) 14(20.9%) 14(20.9%)

% outliers >log1.0c/ml 0 2(2.98%) 3(4.47%)


Mean (%) similarity 99% 100.2% 102.8%

SD (%) similarity 4.92% 4.75% 5.22%

% CV similarity 4.97% 4.71% 5.08%

2

(c) CAP/CTM HIV-1 versus NucliSENS HIV-1, RealTime HIV-1 and CAP/CA 3

CAP/CTM HIV-1 NucliSENS HIV-1vs RealTime HIV-1 CAP/CA HIV-1

Linear Regression

Equation y=0.988x+0.178 y=1.009x+0.08 y=1.004x+0.335

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P-value on intercept 0.452 0.662 0.102

R2 0.857 0.908 0.893

Bland-Altman

Bias in copies/ml -0.126(-0.225;-0.027) -0.124(-0.208;-0.046) -0.352(-0.438;-0.267)

Bias in IU/ml 0.096(-0.002;0.195) -0.136(-0.215;-0.057) 0.16(0.078;0.248)

SD 0.406 0.323 0.349



% outliers >log0.5c/ml 18(26.9%) 7(10.5%) 25(37.3%)

% outliers >log1.0c/ml 0 2(2.98%) 3(4.47%)


Mean (%) similarity 101.5% 101.4% 104%

SD (%) similarity 4.86% 3.86% 4.23%

% CV similarity 4.79% 3.81% 4.07%

1

(c) CAP/CA HIV-1 versus NucliSENS HIV-1, CAP/CTM HIV-1 and RealTime HIV-1 2

CAP/CA HIV-1 NucliSENS HIV-1vs CAP/CTM HIV-1 RealTime HIV-1

Linear Regression

Equation y=0.946x+0.038 y=0.889x+0.191 y=0.937x+0.084

P-value on intercept 0.858 0.323 0.697

R2 0.858 0.893 0.883

Bland-Altman

Bias in copies/ml 0.226(0.137;0.316) 0.35(0.267;0.437) 0.228(0.137;0.318)

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Bias in IU/ml -0.067(-0.157;0.022) -0.16(-0.249;-0.079) -0.30(-0.39;-0.21)

SD 0.366 0.349 0.371



% outliers >log0.5c/ml 14(20.9%) 25(37.3%) 22(32.8%)

% outliers >log1.0c/ml 3(4.47%) 3(4.47%) 0


Mean (%) similarity 97.8% 96.6% 97.8%

SD (%) similarity 4.02% 3.57% 4.35%

% CV similarity 4.11% 3.7% 4.45%

1 2

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Table 3: Assay characteristics for a one-shift result reporting cycle, not including daily maintenance and 1

consumable loading and reagent preparation. 2

Assay Number of

controls

Extraction capacity for one

instrument.

Amplification and detection Number of reportable

patient results/day

Number

of staff

CAP/CA 3/21 samples 1 Ampliprep:

1 run = 21 samples = 2hrs

{2nd

run = 21 samples = 1 hour

3rd run = 21samples = 1hour}&

1 Amplicor (manual mastermix):

1 run = 21 patient samples = 6

hours

21 patient samples (after

hour result reporting)

2

CAP/CTM 3/21 samples 1 Ampliprep:

1 run = 21 samples = 2hrs

2nd run = 21 samples = 1 hour

1 Taqman 48*:

1 run = 42 patient samples = 4

hours

45 patient samples 1

NucliSENS 3/21 samples*

1 EasyMag:

6 runs (21 samples/run) = 4hours

(40mins/run) = 126 samples

1 EasyQ analyser:

3 runs, 42 patient samples each

run = 126 patient samples = 3

hours


Real Time 3/93samples

1 m2000sp:

1 run = 93 samples = 3.5hrs

1 m2000rt (automated mastermix):

1 run = 93 samples = 3.5hrs


*This is laboratory specific. This value is per Johannesburg protocol. & Additional runs can be prepared within same shift, but not amplified/detected within 3

same day4

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1

2

3

4

(a) 5

(b) 6

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(c) 1

(d) 2

Figure 1(a,b,c,d): Bland Altman Analysis scatter plots of the comparison of all assays to 3

each other. The vertical axis is the difference between the assays. The horizontal axis is 4

the average log transformed values of all assays represented as copies/ml. The assay 5

names are given on each plot. 6

7

8

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