The stability of HIV-1 nucleic acid in whole blood and improved detection of HIV-1 in alternative specimen types when compared to Dried Blood Spot (DBS) specimens

Journal of Virological Methods 261 (2018) 91–97

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Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet

The stability of HIV-1 nucleic acid in whole blood and improved detection of HIV-1 in alternative specimen types when compared to Dried Blood Spot (DBS) specimens

T

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Cheryl Jenningsa, , Brian Hartyb, Salvatore R. Sciannaa, Suzanne Grangerb, Amy Couzensc, Daniel Zaccaroc, James W. Bremera a b c

Rush Medical College, Department of Microbial Pathogens and Immunity, Chicago, IL, USA New England Research Institute, Boston, MA, USA RTI International, Research Triangle Park, NC, USA

A R T I C LE I N FO

A B S T R A C T

Keywords: HIV-1 stability HIV-1 detection HIV-1 DBS

Background: Commercially-available kits for HIV-1 detection include instructions for detecting HIV-1 in plasma and DBS, but don’t support other specimen types. Objectives: Show quantitative stability of HIV-1 total nucleic acid (TNA) in blood and improved HIV-1 detection in alternative specimen types. Study design: Whole blood and DBS specimens, tested as part of an external quality assurance program for qualitative HIV-1 detection, were used to evaluated error rates (false negative [FN], false positive [FP] and indeterminant [IND] results) across assays (internally developed [IH], Roche Amplicor [RA], and Roche TaqMan Qual [TQ]) and specimen types (frozen whole blood [BLD], DBS and cell pellets [PEL]). A modified Roche TaqMan HIV-1 assay was used to quantify HIV-1 TNA. Results: Significantly higher error rates were noted in DBS across all of the assays (4% vs. 0% for DBS and PEL, IH, p = 0.005; 4% vs. 0.1% for DBS and PEL, RA, p < 0.001; 10% vs. 1% for DBS and PEL or BLD, TQ, p < 0.001). HIV TNA concentration is stable in BLD (day 1 vs. day 10, p = 0.39) and higher than DBS (p < 0.001). Conclusions: Transporting refrigerated whole blood for centralized processing into alternative specimen types will improve the sensitivitiy of HIV-1 detection in samples with low virus loads.

1. Background The Roche Amplicor® HIV-1 DNA Test, v1.5 (RA) was a commonly used commercially available assay for detecting HIV-1 DNA in cell pellet (PEL) samples and dried blood spots (DBS) around the world (Bogh et al., 2001; Fischer et al., 2004; Germer et al., 2006; John et al., 2012; Lambert et al., 2003; Kebe et al., 2011; Nsojo et al., 2010). The kit offered a whole blood lysis buffer that removed red blood cells containing hemoglobin, which can inhibit PCR, and provided a simple and inexpensive way to create PEL specimens that were frozen and batch tested. The kit utilized an efficient extraction buffer that contained proteinase K, which digested the cellular material and released the HIV-1 proviral DNA for detection by PCR. Batch sizes with the RA kit could contain as few as eight samples without wasting kit components. The Roche COBAS® AmpliPrep/COBAS® TaqMan® HIV-1 Qual

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(TQ) Test, the Abbott RealTime HIV-1 Qual (AQ) assay, and the GenProbe® APTIMA HIV-1 Qualitative (GQ) Assay have replaced the RA assay for qualitative detection of HIV-1. The TQ assay detects HIV-1 total nucleic acids (TNA), the AQ detects both HIV-1 DNA and HIV-1 RNA but at variable frequencies, and the GQ assay only detects HIV-1 RNA. The TQ assay includes DBS and plasma sample options for testing with analytical sensitivities of 20 and 300 copies/mL for plasma and DBS specimens, respectively. The AQ assay includes DBS and plasma options for testing with reported analytical sensitivities of 80 and 2500 copies/mL for plasma and DBS samples, respectively. The DBS sensitivity in both AQ and TQ was established by testing DBS samples produced from free virus particles diluted in HIV-negative whole blood. The GQ assay includes plasma options for plasma samples with a reported analytical sensitivity of 30 copies/mL, though the feasibility of using DBS for the detection of HIV-1 with GQ has been reported (Nelson

Corresponding author. E-mail address: [email protected] (C. Jennings).

https://doi.org/10.1016/j.jviromet.2018.08.009 Received 30 April 2018; Received in revised form 7 August 2018; Accepted 8 August 2018 Available online 17 August 2018 0166-0934/ © 2018 Elsevier B.V. All rights reserved.

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Fig. 1. For proficiency testing, whole blood is collected and aliquoted into coded panels (whole blood or DBS) and shipped under refrigerated or ambient conditions to testing laboratories. For internal stability testing, the whole blood was processed into specimen types over multiple days and then batch tested using the quantitative mRTv2 assay. For ongoing internal stability testing whole blood is frozen on day 1 and 10 of collection and batch tested using the mRTv2 assay.

for batch testing. The use of PEL specimens for detecting HIV-1 nucleic acid testing is also important, not only because these specimens exist in specimen repositories but also because they can be used for the quantitation of cell-associated HIV-1 nucleic acids (Pasternak et al., 2013). The NIAID Virology Quality Assurance (VQA) Laboratory currently pairs the quantitative Roche COBAS® AmpliPrep/COBAS® TaqMan® HIV-1 assay with the TQ off-board extraction (mRTv2) for quantifying the total HIV-1 nucleic acids in DBS, BLD and PEL. Previous studies conducted by the VQA demonstrated that whole blood can be held up to 10 days under refrigerated conditions prior to processing into PEL for testing with the RA assay (Jennings et al., 2005); stability was determined by doing 10 fold serial dilutions of the extracted nucleic acid to quantitate the loss of HIV DNA over time. The use of a quantitative assay to monitor HIV nucleic acid stability, such as the mRTv2 would provide the ability to quantitate differences in HIV-1 TNA across specimen types.

et al., 2014) with a 96.5% sensitivity in samples with plasma viral loads > 300 copies/mL. Although these assays offer many advantages, such as full automation, total HIV-1 nucleic acid detection, and high sample throughput, there are some limitations which include the need for larger batch sizes for testing and the limitation of using only plasma or DBS options defined in the package insert. While whole blood used to be an option for TQ for the version 1 kit, it was removed from the version 2 kit. Plasma can be frozen; however, the use of this sample type will only permit the detection of HIV-1 RNA, which can result in the reporting of false negative results, especially in specimens collected from infants exposed to antiretroviral treatment for prevention of mother-to-child transmission (Burgard et al., 2011; Connolly et al., 2013). Dried blood spots (DBS) are appealing from a cost and handling perspective, but assay sensitivity is much lower in DBS. Even though the volume of blood dried on a DBS spot is small (75 uL), it does contain both plasma virus and cell-associated HIV-1 nucleic acid, so it has the potential to provide more targets for detection in AQ and TQ assays. The problem associated with DBS is ensuring the plasma and cells are eluted from the paper so that both free plasma virus and cell-associated nucleic acids can be detected. Whole blood (BLD), on the other hand, is an attractive alternative specimen, because it requires no elution of the specimen from the paper prior to extraction and can be tested using slightly larger volumes (this study evaluated 0.1 mL) while staying within reasonable volumes for use with pediatric samples (Piwowar-Manning et al., 2008). In laboratories with smaller testing volume, freezing whole blood (BLD, −70 °C) is necessary to collect a sufficient number of samples needed

2. Objectives The purpose of this study was to use data from a multicenter HIV-1 nucleic acid proficiency testing program to compare error rates (false negative [FN], false positive [FP], and indeterminate [IND] results) in HIV-1 detection across the specimen types, donors and assays. An internal study conducted by the VQA was also performed to evaluate the stability of HIV-1 in whole blood stored under refrigerated conditions out to 10 days prior to processing into intermediate specimen types (dried blood spots [DBS], whole blood cell pellets [PEL], frozen whole 92

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each round of testing were included in the analysis of each panel. Data generated using different assays in the same laboratory were treated as separate data sets and analyzed separately. Laboratories were instructed to process the whole blood using the same validated method used for processing local clinical samples. Assays included in this evaluation were the RA, TQ, and an IH assay (Ngo-Giang-Huong et al., 2008). Since the testing was conducted over several years, some laboratories submitted data using multiple assays and multiple specimen types. The laboratories that participated in the DBS proficiency testing panel also participated in the whole blood rounds.

blood [BLD]) that were frozen prior to batch testing, and to quantitate HIV-1 TNA across specimen types. Example data from ongoing monitoring of whole blood stability is also provided as evidence that HIV-1 TNA concentration is stable in BLD that is refrigerated for up to 10 days prior to freezing or testing. 3. Study design (Fig. 1) 3.1. Specimen handling and shipping for proficiency testing Prior to collecting whole blood units for use in creating panels for qualitative HIV-1 nucleic acid proficiency testing, samples from donors enrolled in the VQA Program (Jackson et al., 1993) were screened to confirm their HIV-1 serostatus, plasma viral load and CD4 T-cell counts. HIV-1 RNA testing (plasma) was done during a screening visit prior to any blood donation using a range of viral load assays over time; a range of results may be reported because some donors gave blood across multiple panels. The assays used for testing include UltraSensitive Roche Amplicor HIV-1 Monitor Test, Abbott RealTime HIV-1 RNA Test, or Roche COBAS AmpliPrep COBAS TaqMan HIV-1 RNA test (RTv2). HIV-1 negative donors screened using serology testing, no HIV-1 RNA was performed (ND). Citrate-phosphate-dextrose (CPD) anticoagulated whole blood units were collected from HIV-infected and HIV-uninfected donors and aliquoted into coded panels. The panel samples were shipped to international laboratories under refrigerated conditions using refrigerated gel packs, and to domestic laboratories under ambient conditions using commercial courier services. The collection, processing and shipment of whole blood was performed in one day, but number of days for receipt and processing of the blood varied, ranging from 1 to 10 days. The proficiency testing panel configurations were validated by the VQA laboratory using commercially available HIV-1 nucleic acid assays. A portion of the whole blood was also held overnight under ambient conditions and processed into DBS panels the following day. Fifty (and later 75) microliters of whole blood were spotted onto four spots of appropriately labeled Whatman 903 cards, dried overnight, then placed in a glassine bag and placed into a gas-impermeable bag with desiccants and a humidity indicator (Garcia-Lerma et al., 2009; Mitchell et al., 2008; Youngpairoj et al., 2008). Blood from one donor was aliquoted per card. Dried blood spot panels were packaged as panels and stored at -70 °C until the panels were shipped to testing laboratories (typically 4–8 weeks). Shipping of DBS specimens was done under ambient conditions; panels were thawed, humidity indicators were checked and additional desiccants were added as needed. Laboratories were instructed to store DBS panels at 2–8 °C or frozen at −20 °C or colder until tested. Regardless of storage conditions, humidity indicators were checked prior to testing and humidity exposure was documented. Bags containing DBS cards were not opened until the panels were equilibrated to room temperature to prevent the influx of warm, humid air. Dried blood spot extractions and HIV-1 nucleic acid amplification and detection were done according to the receiving laboratory’s internal procedures. Information concerning the extraction method, amount of spot extracted, and assays used for testing, and the HIV-1 nucleic acid testing result for each sample were submitted to the VQA for analysis.

3.3. Specimen handling for whole blood, cell pellet, and DBS stability evaluations Whole blood from HIV-infected and HIV-uninfected donors collected for a round of HIV-1 nucleic acid proficiency testing was stored by the VQA at 4 °C for a total of 10 days. On days 1, 3, 6, 7, 8, 9, and 10, whole blood was used to generate three derivatives: DBS, PEL and BLD. The DBS samples were created in the same manner described above and frozen until batch tested. The PEL samples were created by washing 0.5 mL of whole blood with the Roche Red Blood Cell Lysis Buffer according to the product insert (Sigma-Aldrich, USA, Cat. No. 118 143 89 001). The BLD samples were created by adding 0.1 mL of whole blood to a 1.8 mL cryovial. All intermediate specimen types were stored at −70 °C until tested. At the time of testing, specimens from all time points, within a specimen type, were thawed, extracted, and assayed in one batch to minimize assay variability. 3.4. Specimen handling for ongoing internal stability testing of whole blood Ongoing monitoring of HIV-1 TNA stability in whole blood specimens used for proficiency testing is evaluated by the VQA laboratory by comparing quantitative total HIV-1 nucleic acid in BLD specimens after 1 and 10 days of storage under refrigerated conditions; data from six additional rounds of testing were provided in this evalutation. 3.5. Estimating error rate percentages and quantifying total HIV-1 nucleic acid in BLD Error types included FN, FP and IND results. Error rates were calculated by taking the total number of errors divided by the total number of results for a given specimen and multiplying by 100 to generate a percentage. The expected result for each sample was based on consensus results obtained during the testing of the whole blood panels; indeterminate results were either based on laboratory-defined criteria or discordant results reported for replicate testing of the same sample. The amount of total nucleic acid in a 0.1 mL volume of EDTA whole blood was quantified by coupling the “off-board” extraction procedure from the TQ kit with the “on-board” lysis and amplification/detection of the quantitative Roche RTv2 kit (mRTv2). Results obtained for this testing were reported as total nucleic acid copies/0.1 mL BLD. For comparisons of error rates, Fisher’s Exact Test was employed to compute p-values. To compare continuous values (e.g., HIV-1 TNA), mixed linear models using Day (1 vs. 10) or sample type (BLD vs DBS) as a fixed factor and panel (209 through 214) as a random factor. All analyses were conducted using SAS statistical software v9.3.

3.2. Testing laboratories for VQA Qualitative HIV-1 DNA proficiency testing

4. Results The data for this study were generated by laboratories participating in the VQA Qualitative HIV-1 DNA whole blood and DBS proficiency testing programs from May 2008 through October 2014. One to 3 data sets were submitted by 6–13 laboratories for a total of 8–16 data sets per round of DBS testing; 1–2 data sets were submitted by 25–34 laboratories for a total of 5–19 data sets per round of whole blood testing. Four rounds of testing per year were conducted with each laboratory receiving a minimum of 2 challenges per year. All data submitted for

4.1. DBS proficiency testing Table 1 shows the donor characteristics for the samples used for the DBS proficiency testing. HIV-infected donors gave blood at different stages of clinical HIV-1 infection; CD4/CD8 ratios ranged from 0.1 to 1.0 in the HIV-infected donors and HIV-1 viral loads ranged from undetectable (TND) to 719,000 copies/mL. Frozen whole blood (BLD) 93

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Table 1 Donor characteristics. Donor ID

Expected Result1

# Rounds Donor Gave Blood2

CD4 Abs

CD8 Abs

CD4/CD8 Ratio

HIV-1 RNA3 cp/mL

Data generated during donor screening visit for each round where blood was donated Donor# Donor# Donor# Donor# Donor# Donor# Donor# Donor# Donor# Donor# Donor# Donor# Donor# Donor# Donor# Donor# Donor# Donor#

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

POS POS POS POS POS POS NEG NEG POS POS POS POS POS POS POS POS NEG POS

8 9 8 6 8 4 11 11 1 3 2 6 2 6 6 1 2 1

641 - 872 526 - 1,037 121 - 246 180 - 216 398 - 957 137 - 333 116 - 598 636 - 1206 846 81 - 116 344 - 348 137 - 384 281 - 311 479 - 630 27 - 180 416 1,076 - 1,115 386

860 - 1,122 717 - 1,176 196 - 517 571 - 768 859 - 1,526 970 - 1,164 410 - 792 180 - 412 1,354 212 - 410 885 - 887 259 - 599 1,178 - 1,244 883 - 1,267 221 - 616 932 310 - 340 671

0.7 0.6 0.5 0.3 0.5 0.1 0.3 2.6 0.6 0.3 0.4 0.5 0.2 0.4 0.1 0.4 3.3 0.6

-

1.0 0.7 0.7 0.4 0.9 0.3 0.8 4.1

TND TND - 50 TND - 50 13,800 - 61,020 TND - < 50 < 50 - 107,394 ND ND < 50 250 - 684 TND < 50 - 19,100 147 - 224 TND - 50 471 - 719,000 TND ND 51

- 0.5 -

1.0 0.3 0.6 0.6

- 3.5

HIV-1 TNA cp/ 0.1 mL BLD4

113 940 265 7,286 497

Error Rate5 # Errors / Total

%

57/170 9/236 26/172 2/161 3/181 0/88 3/280 2/202 0/20 3/82 3/38 3/134 0/40 5/138 1/144 1/28 1/52 1/42

34 4 15 1 2 0 1 1 0 4 8 2 0 4 1 4 2 2

1 Blood from donors collected across panels; expected result was based on consensus results obtained during whole blood proficiency testing within a panel. POS = HIV-1 positive, NEG = HIV-1 negative. 2 A range of plasma viral loads is provided because some donors gave blood across mulitple rounds; screening visits must be conducted prior to each blood donation (Section 3.1). 3 HIV-1 RNA testing (plasma) was done during a screening visit prior to any blood donation using a range of viral load assays over time; a range of results across screening visits are reported. The assays used for testing include UltraSensitive Roche Amplicor HIV-1 Monitor Test, Abbott RealTime HIV-1 RNA Test, or Roche COBAS AmpliPrep COBAS TaqMan HIV-1 RNA test (RTv2). TND = Target Not Detected, < 50 = qualitatively detected HIV-1 RNA by Roche Monitor. HIV-1 negative donors screened using serology testing, no HIV-1 RNA was performed (ND). 4 Total HIV-1 nucleic acid (TNA) is the quantitative result obtained from frozen 0.1 mL EDTA Blood using the modified RTv2 assay using blood donated for use in the panel for a single collection. The result is the average of two replicates from a single time point. Blank cells indicate testing was not performed for any visit. 5 The error rate (%) is the total number of errors divided by the total number of specimens tested x 100 (for all specimen types combined by donor).

testing was performed on samples from 5 donors. HIV-1 total nucleic acid (TNA) in 0.1 mL of whole blood ranged from 113 to 7286 copies/ 0.1 mL BLD. Four of these 5 HIV-infected donors had undetectable or very low plasma viral loads (< 50 or 50 copies/mL), and HIV-1 TNA in those samples ranged from 113 to 940 copies/0.1 mL BLD; the one remaining donor had higher plasma viral loads (13,800–61,020 copies/ mL) which corresponded with a higher TNA result (7286 copies/0.1 mL BLD). Thirty-four percent of all the results reported (51/170) across 8 different rounds of testing for samples created using blood from donor #1 were scored as errors in proficiency testing (TNA = 113 cp/0.1 ml BLD), and 15% of all of the results reported (26/172) across 8 rounds of testing for samples created using blood from donor # 3 were scored as errors (TNA = 265 copies/0.1 mL BLD); error rates in specimens derived from other donors ranged from 0% to 8%; HIV-1 TNA concentrations in 0.1 mL BLD were not available for all donors. Table 2 compares the error rates data generated for all specimen types by assay. The number of samples tested includes the total number of samples tested across all laboratories using the same testing parameters. Abbott Qual data were excluded because only results for DBS were used for testing. The error rate was 4% vs. 0% for DBS and PEL in IH assays (p = 0.005), 4% vs. 0.1% for DBS and PEL in RA data (p < 0.001), and was 10% vs. 1% for DBS and PEL or BLD samples in TQ data (p < 0.001); the error rates reported in the whole blood testing round (PEL or BLD) were all reported for samples derived from the whole blood of donor #1.

Table 2 Frequency and type of error with respect to assay and specimen type. Assay1

Specimen Type2

# Errors3

# Samples Tested4

Error Rate5

Type of Error6

IH IH RA

DBS PEL DBS

6 0 59

144 200 1,456

4% 0% 4%

6 IND

RA TQ

PEL DBS

2 55

2,576 560

0.1% 10%

TQ

PEL, BLD

4

440

1%

51 FN, 5 IND, 3 FP 1 FN, 1 FP 41 FN, 13 IND, 1 FP 3 FN

1

IH = Internally Developed Assay; RA = Roche Amplicor HIV-1 DNA Test, v1.5, TQ = Roche COBAS AmpliPrep COBAS TaqMan HIV-1 Qual. 2 DBS = dried blood spot, PEL = cell pellet, BLD = frozen whole blood. 3 The # errors reported by all laboratories (Sections 3.1 and 3.2) using those testing parameters. 4 The total number of samples tested across all laboratories using the same testing parameters. Abbott Qual data were excluded because no other specimen types were used for testing. 5 The error rate (%) is the total number of errors divided by the total number of specimens tested x 100. The p-values for comparisons within each assay are p = 0.005 (IH), p < 0.001 (RA), and p < 0.001 (TQ). 6 FN = false negative results; FP = false positive results; and IND = indeterminate results were either based on laboratory-defined criteria or discordant results reported for replicate testing of the same sample.

was assigned as the CT value for a false negative result in order to estimate the average CT for tracking stability. No FN results were noted throughout the 10 day stability testing of specimens derived from blood collected for donors #2 and #6. The CT values were 32.0 ± 0.56 for DBS, 31.5 ± 0.68 for PEL, and 31.3 ± 0.96 for BLD samples from donor #2. The CT were 27.4 ± 0.44 for DBS, 29.0 ± 0.69 for PEL,

4.2. Whole blood stability experiments Whole blood stability data are provided in Fig. 2. The average cycle threshold (CT) data for the replicates tested were plotted by specimen type and days of storage at 4 °C for each donor. The TQ assay reports a sample as negative if the CT value is > 39; for this analysis, a CT of 40 94

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Fig. 2. Whole blood from three HIV-infected donors was held at 4 °C for 10 days and aliquots of blood were processed at intervals to create dried blood spot (DBS), whole blood pellets (PEL) or frozen whole blood (BLD). Derivatives were stored at −70 °C until all the time points were processed. Testing was performed using the Roche COBAS AmpliPrep COBAS TaqMan HIV-1 Qual assay; all specimens types were extracted using the DBS procedure (Section 3.3). Note: an arrow indicates the timepoint where one of the duplicates of the DBS derived from blood collected from Donor#1 yielded a false negative result.

was 0.85.

and 26.7 ± 0.28 for BLD samples from donor #6. Three FN specimens were noted in DBS specimens obtained from donor #1 on day 1, 7, and 8; only one replicate of the duplicate DBS tested at each time point was negative. No FN results were noted in any of the other specimen types derived from this donor during the stability evaluation. The CT values for the positive DBS samples 36.1 ± 2.35 for DBS, 34.1 ± for PEL, and from 33.9 ± 1.05 for BLD samples from donor #1. No errors were noted in any of the specimens derived from the HIV-uninfected donor #8 during the stability study (data not shown). Data from ongoing internal validation of VQA proficiency testing panel are provided in Table 3. The HIV-1 TNA in DBS on day 1 after collection ranged from 57 to 9057 copies/spot (75 uL, geometric mean = 994 copies/spot) and corresponding concentration in BLD (0.1 mL) ranged from 303 to 15,972 copies/0.1 mL BLD for the same donors (geometric mean = 3128 copies/0.1 mL BLD, p < 0.001); the fold difference in HIV TNA was 0.32. There were no significant losses in HIV-1 TNA between day 1 (geometric mean = 3128 copies/0.1 mL) and day 10 (geometric mean = 2660 copies/0.1 mL) when looking within BLD specimen type (p = 0.39); the fold change from day 1 to day 10

5. Discussion Data generated for purposes of external quality assessment (EQA) for qualitative HIV-1 DNA or total nucleic acid (TNA) detection in whole blood sample derivatives (frozen whole blood [BLD], cell pellets [PEL]) and dried blood spots (DBS) were used in this evaluation to show how detection of HIV-1 nucleic acids varies across specimen types (DBS, BLD, and PEL). Stability studies also included in this evaluation suggest that transporting whole blood under refrigerated conditions to a central laboratory for processing and storing may yield higher quality specimens than DBS for early diagnosis of HIV-1 infection, especially if the plasma virus load may be reduced due to exposure to antiretroviral treatment to prevent transmission of HIV. Quantitative analysis of BLD vs. DBS derived from the same whole blood sample shows that BLD samples contain 3–20 times more HIV-1 TNA than DBS and suggest variable elution efficiencies for DBS extraction methods. Finally, by comparing EQA data generated from whole blood and DBS panels, this

Table 3 Comparison of HIV-1 TNA concentration in BLD and DBS specimens. TYPE

BLD DBS

HIV-1 TNA copies D11

D10

n=

Geometric Mean

MIN

MAX

n=

Geometric Mean

MIN

MAX

p-value

36 36

3,128 994

303 57

15,972 9,057

36 ND

2,660 ND

216 ND

16,777 ND

0.392 < 0.0013

1 The geometric mean, miminum (MIN), and maximum (MAX) HIV-1 TNA concentration in 36 samples tested at day 1 (frozen whole blood [BLD], dried blood spots [DBS]) and day 10 (BLD) after collection. HIV-1 TNA concentration was measured using a modified Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 RNA assay (Section 3.4). 2 BLD HIV-1 TNA copies/0.1 mL created on the same day of collection (D1) vs. BLD HIV-1 TNA copies/0.1 mL from samples created after ten days of storage (D10); p-value derived from mixed model using day as fixed factor and panel as random factor (interaction of day * panel, p = 0.95); note fold change from Day 1 to Day 10 in BLD= 0.85. 3 BLD HIV-1 TNA copies/0.1 mL for D1 testing vs. DBS HIV-1 TNA copies/spot (0.075 mL) from D1 testing; p-value derived from mixed model using sample type as fixed factor and panel as random factor (interaction of sample type * panel, p = 0.14); note fold difference between DBS vs BLD is 0.32.

95

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manufacturer switched from using two DBS spots for testing to one spot, again suggesting that increasing the blood volume increases inhibition rather than improves sensitivity. For EQA, laboratories use whatever assays and specimen types are validated for use in their laboratories; the VQA has the opportunity to evaluate alternate specimen types since testing is being performed for quality assurance not for clinical management and well characterized specimens are available for evaluation. Improving extraction methods by using heat or Proteinase K with agitation are common ways to improve extraction but if the methods are not supported by the manufacturer’s claims then a laboratory must spend extra time and money to validate the new extraction method. Modifying the specimen type also requires validation but if no additional reagents or equipment are needed to do the testing, the process is simpler. This VQA stability study demonstrated the feasibility of using alternative specimen types (PEL or BLD) for either quantitative (mRTv2) or qualitative (TQ) detection of HIV-1 nucleic acids by Roche TaqMan, and the quantitative results for BLD specimens show that the HIV-1 nucleic acids are stable when stored at −70 °C, even if frozen after 10 days of refrigerated whole blood storage. In DBS proficiency testing, samples generated from blood containing low concentrations of HIV-1 TNA (< 300 copies/0.1 mL BLD) were associated with higher error rates, but the error rates for BLD or PEL was significantly less regardless of the assays used. Clearly the amount of nucleic acid present will impact the sensitivity of the assay and if that concentration is near the limit of detection for that assay then false negative results would be expected. However, starting with the intermediate specimen type that will yield the highest concentration of HIV-1 nucleic acids will ensure the best detection rate for any assay and those optimal specimen types (e.g. BLD and PEL) are not currently validated by the manufacturers of the current assays. The current assays for HIV-1 DNA/TNA testing typically require that blood is processed within 4 days of collection (TQ and Abbott Qual package inserts). The VQA Program previously reported the stability of whole blood prior to processing for HIV-1 DNA testing using the Roche Amplicor HIV-1 DNA test (RA) if the blood was kept under refrigerated conditions prior to processing into PEL for testing (Jennings et al., 2005); and the current study was designed to show similar stability for whole blood tested with the TQ and modified RTv2 assays using multiple intermediate specimen types. In the current study, whole blood was collected by the VQA and held locally for 10 days under refrigerated conditions prior to processing the blood into intermediate specimens for batch testing. Washed whole blood pellets (PEL), DBS, and BLD specimens were created on days 1, 3, 6, 7, 8, 9 and 10 and frozen at −70 °C until tested. Replicate samples, from the same donor and included in each panel, were assayed separately. Sporadic false negative samples in one replicate of the panel were reported on day 1, 7, and 8 in a donor with a low HIV-1 TNA in 0.1 mL BLD (113 copies/ 0.1 mL); the timing of the false negative results suggests the errors were related to the amount of nucleic acid extracted which likely impacted assay sensitivity and was not related to nucleic acid stability. No errors were reported in any of the other specimens created for stability testing and demonstrates that blood can be collected in remote settings and transported in a cooler with gel packs to a central laboratory for processing ensure the best sensitivity as the amount of nucleic acid lost over time will vary by specimen. These data also confirm that PEL and BLD derivatives offer reliable specimen types for the qualitative HIV-1 nucleic acid detection using the TQ and for quantitative HIV-1 TNA using a modified RTv2 assay. Ongoing studies are needed to further optimize extraction methods and to evaluate the quantitative HIV-1 TNA testing in whole blood and cellular samples to determine if these samples can be used to monitor compartment viral loads, and the stability of these samples over time when stored at −70 °C.

study shows that higher error rates (false negative and indeterminate) occur in DBS than in other specimen types, even within the same assay; relatively few false positive results were noted with any assay and were usually associated with sample switching. The relevance of this study to early diagnosis of HIV is that the current assays being used for early diagnosis only provide options for DBS and plasma samples, with the exception of the Cepheid Xpert HIV-1 Qual that offers options for whole blood and provides sensitivity similar to that noted in BLD tested on the Roche COBAS® AmpliPrep/COBAS® TaqMan® HIV-1 Qual (TQ) assay (data not shown); this study shows that the use of BLD is certainly feasible for use with the TQ and mRTv2 assay, and can be held under refrigerated conditions for up to ten days before processing or testing. Since automated assays for detecting HIV-1 TNA such as the TQ are designed for running larger batches of 24 samples, identifying a stable intermediate specimen type that can be stored until sufficient samples are available for testing is very important. The TQ assay is validated for DBS and plasma specimen types, but this study shows that sporadic false negative results can occur, especially in samples with undetectable plasma viral loads, which may be problematic for early infant diagnosis in the setting of prophylactic HIV-1 antiretroviral treatment (Burgard et al., 2011; Connolly et al., 2013) being used to prevent HIV-1 transmission. The use of DBS for virologic testing is more convenient than plasma because it doesn’t need to be frozen and transported on dry ice. In fact, when packaged correctly, DBS can withstand extreme temperatures and even undergo multiple freeze/thaw events and still be used for measuring virologic nucleic acid species such as HIV-1 RNA and HIV-1 DNA, or for performing HIV-1 drug resistance testing (Brambilla et al., 2003; Buckton et al., 2008; Cassol et al., 1992; Garrido et al., 2008; McNulty et al., 2007; Stevens et al., 2008). Humidity, on the other hand, is problematic because it not only permits fungal and bacterial growth, but also hinders the elution of material from the DBS paper (Garcia-Lerma et al., 2009; Mitchell et al., 2008; Youngpairoj et al., 2008). The data submitted for EQA were generated using a variety of extraction and assay combinations including both commercially available and an internally-developed assay (Ngo-Giang-Huong et al., 2008), but since the DBS were created by the VQA using a standardized process, this component of variation did not affect this analysis. For proficiency testing, laboratories processed the whole blood into whatever derivative that was validated by their laboratory: laboratories using the Roche Amplicor (RA) assay used cell pellet (PEL) samples, the laboratory using an IH assay processed the blood into PEL for testing, and laboratories using TQ used either PEL or BLD. A subset of the same laboratories that participated in the whole blood round also participated in the DBS round using the same assays. Therefore, the only difference between rounds was the specimen type and laboratory bias did not contribute to the outcomes noted in this evaluation. Furthermore, since the same blood was used to create DBS and whole blood panels, the consensus results obtained from whole blood testing were used to assess the sensitivity of results for DBS testing. Data generated in this study show that HIV-1 total nucleic acid (TNA) can be 3–20 times higher in 0.1 mL BLD compared to the nucleic acid eluted from a 75 uL spot. Donor characteristics (specifically viral load and HIV-1 TNA in BLD) contributed to the variability in the detection of HIV-1 nucleic acid in DBS samples. The results from this analysis suggest that standardized processing for creating and storing DBS samples are important and that the best detection rates are obtained when a whole spot containing 75 uL of blood is used for extraction (data not shown), and that not all extraction methods have acceptable performance even with a commercial amplification and detection kit. Internal studies conducted by the VQA laboratory has shown that the addition of Proteinase K to the extraction buffer can improve extraction of HIV-1 TNA from cell pellet samples for detection on the Abbott HIV-1 Qual assay, but did not improve the detection of HIV-1 TNA in blood suggesting that the hemoglobin was contributing to the inhibition noted in whole blood testing on that assay (data not shown). In the updated version of the Abbott DBS assay, the 96

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Acknowledgements

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This project described was supported by the Virology Quality Assurance Program (HHSN272201200023C, HHSN266200500044C) from NIAID. The content is solely the responsibility of the authors and does not necessarily represent the official views of NIAID or the National Institutes of Health. Thanks to Ms. Lei Zhang and Mr. Nicolai Arendovich for their assistance in the testing of these samples and to Roche Molecular Systems for supplying kits to perform stability testing. Ms. Suzanne Granger contributed to this work while employed at the New England Research Institute, Inc., Watertown, MA as a VQA contractor. Thanks to Dr. Joseph Fitzgibbon and Dr. Keith Crawdford for their careful review of the manuscript. References Bogh, M., Machuca, R., Gerstoft, J., Pedersen, C., Obel, N., Kvinesdal, B., Nielsen, H., Nielsen, C., 2001. Subtype-specific problems with qualitative amplicor HIV-1 DNA PCR test. J. Clin. Virol. 20, 149–153. Brambilla, D., Jennings, C., Aldrovandi, G., Bremer, J., Comeau, A.M., Cassol, S.A., Dickover, R., Jackson, J.B., Pitt, J., Sullivan, J.L., Butcher, A., Grosso, L., Reichelderfer, P., Fiscus, S.A., 2003. Multicenter evaluation of use of dried blood and plasma spot specimens in quantitative assays for human immunodeficiency virus RNA: measurement, precision, and RNA stability. J. Clin. Microbiol. 41 (5), 1888–1893. Buckton, A.J., Bissett, S.L., Myers, R.E., Beddows, S., Edwards, S., Cane, P.A., Pillay, D., 2008. Development and optimization of an internally controlled dried blood spot assay for surveillance of human immunodeficiency virus type-1 drug resistance. J. Antimicrob. Chemother. 62, 1191–1198. Burgard, M., Blanche, S., Jasseron, C., Descamps, P., Allemon, M.-C., Ciraru-Vigneron, N., Floch, C., Heller-Roussin, B., Lachassinne, E., Mazy, F., Warszawski, J., Rouzioux, C., on behalf of the Agence Nationale de Recherche sur le SIDA et les Hepatites virales French Perinatal Cohort, 2011. Performance of HIV-1 DNA or HIV-1 RNA tests for early diagnosis of perinatal HIV-1 infection during anti-retroviral prophylaxis. J. Pediatr. 160 (1), 60–66. Cassol, S., Salas, T., Gill, J.M., Montpetit, M., Rudnik, J., Sy, C.T., O’Shaughnessy, M.V., 1992. Stability of dried blood spot specimens for detection of human immunodeficiency virus DNA by polymerase chain reaction. J. Clin. Microbiol. 30 (12), 3039–3042. Connolly, M.D., Rutstein, R.M., Lowenthal, E.D., 2013. Virologic testing in infants with perinatal exposure to HIV receiving multidrug prophylaxis. Pediatr. Infect. Dis. J. 32 (2), 196–197 (Letter). Fischer, A., Lejczak, C., Lambert, C., Servais, J., Makombe, N., Rusine, J., Staub, T., Hemmer, R., Schneider, F., Schmit, J.C., Arendt, V., 2004. Simple DNA extraction method for dried blood spots and comparison of two PCR assays for diagnosis of vertical human immunodeficiency virus type 1 transmission in Rwanda. J. Clin. Microbiol. 42 (1), 16–20. Garcia-Lerma, J.G., McNulty, A., Jennings, C., Huang, D., Heneine, W., Bremer, J.W., 2009. Rapid decline in the efficiency of HIV drug resistance genotyping from dried blood spots (DBS) and dried plasma spots (DPS) stored at 37°C and high humidity. J. Antimicrob. Chemother. 64, 33–36. Garrido, C., Zahonero, N., Fernandes, D., Serrano, D., Silva, A.R., Ferraria, N., Antunes, F., Gonzalez-Lahoz, J., Soriano, V., De Mendoza, C., 2008. Subtype variability, virological response and drug resistance assessed on dried blood spots collected from HIV patients on antiretroviral therapy in Angola. J. Antimicrob. Chemother. 61,

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