Evaluation of a Dried Blood and Plasma Collection Device,SampleTanker ®,for HIV Type 1 Drug Resistance Genotyping in Patients Receiving Antiretroviral Therapy

Abstract

Whatman 903 filter paper is the only filter paper that has been used for HIV drug resistance (HIVDR) genotyping in resource-limited settings. In this study, we evaluated another dried blood specimen collection device, termed SampleTanker^® (ST), for HIVDR genotyping. Blood specimens from 123 antiretroviral therapy (ART)-experienced patients were used to prepare ST whole blood and ST plasma specimens; they were then stored at ambient temperature for 2 or 4 weeks. The remaining plasma specimens were stored at −80°C and used as frozen plasma controls. Frozen plasma viral load (VL) was determined using the Roche Amplicor HIV-1 Monitor test, v.1.5 and 50 specimens with VL ≥3.00 log₁₀ copies/ml were genotyped using the broadly sensitive genotyping assay. The medium VL for the 50 frozen plasma specimens with VL ≥3.00 log₁₀ was 3.58 log₁₀ copies/ml (IQR: 3.32–4.11) and 96.0% (48/50) of them were genotyped. Comparing to frozen plasma specimens, significantly lower genotyping rates were obtained from ST whole blood (48.98% and 42.85%) and ST plasma specimens (36.0% and 36.0%) stored at ambient temperature for 2 and 4 weeks, respectively (p<0.001). Nucleotide sequence identity and resistance profile analyses between the matched frozen plasma and ST whole blood or ST plasma specimens revealed high nucleotide sequence identities and concordant resistance profiles (98.1% and 99.0%, and 96.6% and 98.9%, respectively). Our results indicate that with the current design, the ST may not be the ideal dried blood specimen collection device for HIVDR monitoring for ART patients in resource-limited settings.

Introduction

Dried fluid spots, especially dried blood spots (DBS), are simple and inexpensive specimen collection methods and useful for blood collection in resource-limited settings with poor access to diagnostic facilities. The main advantages of DBS specimens are that only a small quantity of blood (50–100 μl) is required to make a spot and that they are easy to obtain, stable for a certain period of time when stored at ambient temperature, considered noninfectious, and can be transported to a reference laboratory at minimal cost using routine courier services.^1

–5 Studies have shown that DBS specimens can be an acceptable alternative to plasma specimens for HIV-1 serology⁶ and molecular assays including routine early HIV-1 diagnosis for infants born to HIV-1-infected mothers.^7
–9 Recent studies have also shown that DBS specimens were comparable to plasma specimens for viral load (VL) and HIV drug resistance (HIVDR) analysis in antiretroviral therapy (ART) patients.^3,10

With the rapid expansion of ART coverage to over 8 million HIV-infected patients and the recently updated WHO recommendation for HIVDR prevention and assessment strategy using DBS specimens,^11,12 the number of HIVDR monitoring surveys is increasing in resource-limited countries. With DBS being the preferred specimen type, there is an increase in the demand for filter papers. Having only one source of recommended filter paper, the Whatman 903, for HIVDR monitoring surveys may lead to shortages if unforeseen manufacturing difficulties occur. Diversifying the type of dried blood collection devices available for HIVDR monitoring surveys could decrease the cost through price competition and increase the availability of dried blood collection devices, thus positively impacting low- and middle-income countries for HIVDR monitoring surveillance programs. We have been conducting a series of studies on evaluations of the suitability of additional dried blood specimen collection devices for HIVDR monitoring surveys in ART patients.¹³ In this article, we describe the results from a study that was designed to evaluate SampleTanker (ST), another type of dried specimen transport device that was developed for storage and transport of air-dried specimens at ambient temperature (Research Think Tank, Inc., Alpharetta, GA) for HIVDR genotyping.^14

–20 This device was applied to collect dried whole blood (termed ST whole blood) and dried plasma (termed ST plasma) specimens from a cohort of ART-experienced patients in San Pedro Sula and Tegucigalpa, Honduras for HIVDR genotyping. The purpose of the project was to evaluate the genotyping efficiency of ST whole blood and ST plasma specimens from ART-experienced patients when stored at ambient temperature.

Materials and Methods

Patient clinical characteristics

Between February and March 2008, we obtained remnant blood specimens from 123 patients attending two ART clinics in Tegucigalpa and San Pedro Sula, Honduras. All the patients were receiving ART and the mean time duration on ART was 2.5 years [interquartile range (IQR): 1–4]. The ART regimens were mainly composed of nucleoside reverse transcriptase inhibitors (NTRIs) and nonnucleoside reverse transcriptase inhibitors (NNRTIs) in three drug combinations with the exception of seven patients who were on protease inhibitor (PI)-based second-line regimens.

Sample preparation and storage

For the preparation of ST whole blood specimens 1 ml of whole blood specimens from each patient was loaded onto each ST matrix and allowed to dry in a biosafety cabinet overnight. After drying, the ST whole blood specimens were sealed tightly with desiccant-containing ST transport and storage tubes according to the manufacturer's instructions (Research Think Tank, Inc., Alpharetta, GA). For the preparation of ST plasma specimens, the remaining blood specimens were centrifuged at 1500×g for 20 min and 1 ml of plasma was then loaded onto each ST matrix, and the ST plasma specimens were further processed following the procedure used for the ST whole blood specimens. The remaining plasma specimens were then stored at −80°C and used as frozen plasma controls. To ensure the quality of specimens all the blood specimen preparation procedures were carried out within 6 h of blood collection. After preparation, all the STs were stored at the collection sites at ambient temperature (20–35°C) for 2 or 4 weeks before shipping to the WHO-accredited Specialized Drug Resistance Laboratory (SDRL) at the International Laboratory Branch, Centers for Disease Control and Prevention (CDC), Atlanta, GA by express mail within 2 days at ambient temperature with the exception of frozen plasma specimens, which were shipped on dry ice. All the specimens were stored at −80°C before testing at CDC. The study protocol was approved by the local institutional review board, and the anonymous genotyping tests performed at CDC were deemed as nonhuman subject research by the Associate Director for Science (ADS) at the Center for Global Health, CDC.

Viral load measurement

Roche Amplicor HIV-1 Monitor test, version 1.5 (Roche Molecular Systems Inc., Branchburg, NJ) was used to determine VL for the frozen plasma specimens following the manufacturer's instructions. When 200 μl of plasma was used for testing, the assay had a lower limit of detection of 2.60 log₁₀ copies/ml.

Reconstitution of ST whole blood and ST plasma specimens, nucleic acid extraction and HIVDR genotyping

Before reconstitution, STs were removed from the freezer and allowed to equilibrate to room temperature; the color of the desiccant in the transport indicator capsules was then reviewed and only those that maintained blue color in the desiccant, indicating no moisture contamination, were used for testing. To elute ST whole blood or ST plasma specimens, we followed the manufacturer's instructions. In brief, each unit of the ST matrix was incubated with 1.175 ml of rehydration buffer for 30 min at room temperature. Reconstituted plasma or whole blood was then recovered using the ST recovery kit following the manufacturer's instructions and in general 1 ml of eluent was recovered. For nucleic acid extraction, frozen plasma, ST whole blood, or ST plasma specimens (200 μl) were added to 2 ml of NucliSENS lysis buffer and incubated for 10 min at room temperature. Nucleic acid was then extracted from all the specimens using the NucliSENS EasyMAG automated extraction system (Biomerieux, Durham, NC) according to the manufacturer's instructions. Nucleic acid was eluted in 50 μl of NucliSENS Extraction Buffer 3 and 10 μl of the extract was used for genotyping or stored at −80°C until use.

HIVDR genotyping was performed using the broadly sensitive and low cost in-house genotyping assay.^21,22 This assay has been validated and extensively used for HIVDR genotyping with specimens collected from resource-limited countries and had an analytical genotyping sensitivity of ≥95% when VL was ≥3.00 log₁₀ copies/ml of plasma.^3,10,22,23 Briefly, a 1,084-base pair segment of the 5′ region of the pol gene was generated by reverse transcriptase polymerase chain reaction (RT-PCR) followed by nested PCR. In the case of a failed first RT-PCR attempt, RT-PCR was repeated using a rescue primer following the validated standardized procedure.²² The confirmed PCR product was purified, sequenced using the BigDye Terminator v.3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA), and analyzed on the ABI Prism 3730 Genetic Analyzer (Applied Biosystems, Foster City, CA). The sequence analysis was conducted using ChromasPro software version 1.42 (Technelysium Pty Ltd, Tewantin, Australia) and the drug resistance profile was determined using the Stanford drug resistance database algorithm.^24
–26

Statistical analyses

Continuous data are presented as median and IQR. Rates are given with 95% confidence intervals (CIs). Fisher's exact test was used to compare HIVDR genotyping efficiencies between the groups. All tests were two-sided and a p-value of <0.05 was considered statistically significant. SPSS software version 17.0 (SPSS Inc., Chicago, IL) was used for all the analyses.

Results

Clinical and virological characteristics of the patients

In this study, 123 ART patients were included, consisting of 51 females and 72 males. The ART regimens prescribed were mainly composed of NTRIs and NNRTIs in three drug combinations. Sixty-four percent (79/123) of the patients were on lamivudine/zidovudine/efavirenz (3TC/AZT/EFV), 22.8% (28/123) were on lamivudine/stavudine/nevirapine (3TC/D4T/NVP), 7.3% (9/123) were on other three-drug combinations, while 5.7% (7/123) were receiving boosted-protease inhibitor (PI) based regimens.

Fifty-four (43.9%) of the 123 frozen plasma specimens had VL (VL <2.60 log₁₀ copies/ml) below the lower limit of detection, 15.4% (19/123) between 2.60 and <3.00 log₁₀ copies/ml, and the remaining 40.7% (50/123) ≥3.00 log₁₀ copies/ml. For those 69 specimens with detectable VL, the median VL was 3.47 log₁₀ copies/ml (IQR: 2.97–3.95). Genotyping was only performed on the 50 specimens with frozen plasma VL ≥3.00 log₁₀ copies/ml following the WHO recommendation for HIVDR monitoring surveys²⁷ as well as with the consideration of the analytical genotyping sensitivity of the broadly sensitive genotyping assay (≥95% at VL ≥3.00 log₁₀ copies/ml).^3,10,23 The median VL for these specimens was 3.54 log₁₀ copies/ml (IQR: 3.32–4.11).

Genotyping efficiency of ST whole blood and ST plasma specimens in the HIV-1 pol gene region

All the 50 frozen plasma specimens with VL ≥3.00 log₁₀ copies/ml were genotyped and 48/50 (96.0%) were successful. However, when genotyping was conducted with the matched ST plasma specimens stored at ambient temperature for 2 and 4 weeks, we were able to genotype only 18 of them at both time points (Table 1). Genotyping analyses of the matched ST whole blood specimens stored at ambient temperature for 2 and 4 weeks appear to give better results: 24 at 2 weeks and 21 at 4 weeks, respectively (Table 1). However, compared to frozen plasma specimens, genotyping efficiencies for ST plasma specimens stored for 2 and 4 weeks (both at 36.0%, 95% CI: 23–49; p<0.001) and ST whole blood specimens stored for 2 weeks (49.0%, 95% CI: 35.0–62.9; p<0.001) and 4 weeks (42.9%, 95% CI: 28.9–58.9; p<0.01) were statistically significantly lower than that for frozen plasma specimens (Table 1). The differences seen in genotyping efficiencies between ST plasma and ST whole blood specimens stored at 2 and 4 weeks were statistically insignificant (p=0.48 and p=0.19, respectively). In addition, we did not notice the correlation between genotyping successful rates and VL levels. For those few specimens we noticed insufficient reconstitution process due to the surface tension of the ST matrix device and the viscosity of the whole blood, we also did not see the reduced genotyping rates when compared to the rest of the ST whole blood specimens.

Table 1.

Dried Fluid SampleTanker Genotyping Efficiency and Pairwise Nucleotide Sequence Identity Compared to Frozen Plasma Specimens with HIV-1 RNA≥3 Log₁₀ Copies/ml

		Dried plasma SampleTanker		Dried whole blood SampleTanker
	Plasma	2 weeks storage at AT	4 weeks storage at AT	2 weeks storage at AT	4 weeks storage at AT
Genotyping rate % (No.)	96.0 (48/50)	36.0 (18/50)^*	36.0 (18/50)^*	49.0 (24/49)^*	42.9 (21/49)^*
Nucleotide identity % (95% CI)	NA	99.7 (97.2–102.2)	98.2 (92.1–104.3)	97.9 (92.2–103.6)	98.2 (92.5–103.9)
DRM concordance Rate % (95% CI)	NA	98.9 (94.0–103.8)	98.9 (94.0–103.8)	96.5 (89.2–103.8)	96.6 (88.8–104.4)

p<0.001 (Fisher's exact test).

AT, ambient temperature; NA, not applicable; CI, confidence interval; DRM, drug resistance mutations.

Nucleotide sequence identity and HIV-1 drug resistance mutation profile analyses

Comparative analysis of nucleotide sequence identity between the matched frozen plasma and ST whole blood or ST plasma specimens revealed high sequence identities despite having significantly reduced genotyping rates (Table 1). The sequence identities for ST plasma specimens stored for 2 and 4 weeks were 99.7% (95% CI: 97.2–102.2) and 98.2% (95% CI: 92.1–104.3), and for ST whole blood specimens they were 97.9% (95% CI: 92.2–103.6) and 98.2% (95% CI: 92.5–103.9), respectively (Table 1).

We next analyzed the HIVDR profiles according to the HIValg (HIVdb version 6.2.0) deployed by the Stanford University HIV Drug Resistance Database to see whether the differences of HIVDR mutations identified between frozen plasma and ST whole blood or ST plasma specimens had any clinical significance. For these analyses, we included only those specimens that had paired frozen plasma and ST plasma or ST whole blood specimens including 18 paired frozen plasma and ST plasma specimens stored at ambient temperature for 2 and 4 weeks, and 24 and 21 paired frozen plasma and ST whole blood specimens stored at ambient temperature for 2 and 4 weeks, respectively.

Analyses showed that the overall concordance between frozen plasma and ST plasma specimens was high (98.9%, 95% CI: 94.0–103.8). We identified 87 drug resistance-associated mutations (DRMs) in the protease and RT regions from the frozen plasma specimens. These DRMs were also detected in the ST plasma specimens with the exception of one secondary mutation (P225H in specimen HN047) in the RT region. This mutation is known to increase EFV resistance when combined with K103N mutation. However, in this particular case, it did not alter the clinical outcome because of the presence of L100I in both the frozen plasma and ST plasma specimens, which had already induced high-level resistance to EFV. In addition, three DRMs (K70R, L74LV, and V179IT) were identified from only four ST plasma specimens (HN032, HN047, HN100, and HN117). It is known that K70R induces intermediate-level resistance to zidovudine (AZT) and low-level resistance to stavudine (d4T) and tenofovir disoproxil fumarate (TDF), while L74V causes high- and intermediate-level resistance to didanosine (ddI) and abacavir (ABC). However, due to the presence of the K219E mutation, which induces AZT and d4T resistance in both specimen types for the patient who harbored the K70R mutation, the presence of the K70R mutation did not have a clinical impact for this patient. V179T is a rare mutation that may contribute to reduce etravirine (ETR) susceptibility when combined with other NNRTI mutations, while V179I is a common polymorphism. Their clinical significance in this patient appears to be minimal.

Similarly, analysis of resistance profiles between 24 and 21 paired frozen plasma and ST whole blood specimens stored at ambient temperature for 2 and 4 weeks, respectively, revealed that the overall concordance rate was 96.6% (95% CI: 89.0–104.1). Eighty-five DRMs were identified in frozen plasma specimens while 82 of them were found in the ST whole blood specimens. Three DRMs from two patients (HN103 harbored K238N and HN117 had M184V and G190S) were missing in the ST whole blood specimens. M184V causes high-level resistance to 3TC and FTC and low-level resistance to DDI and ABC, while G190S induces high-level resistance to nevirapine (NVP) and efavirenz (EFV). As the result of these missed mutations in the ST whole blood specimen in this patient and if this patient is monitored using an ST whole blood specimen, it would have led to an incorrect prediction of the resistance profile. On the contrary, for the second patient missing the K238N in the ST whole blood specimen it would not have altered clinical drug susceptibility as the specimen from this patient also harbored the K103N mutation, which induces high-level resistance to the same drugs (EFV and NVP). In addition, we also identified two secondary mutations (T69A and V179DT) in the ST whole blood specimens that were absent from the frozen plasma specimens. While the impact of T69A on NRTIs is unknown, V179DT induces low-level resistance to NNRTIs when combined with other NNRTI-associated mutations. Because the L100I, K103R, and K103N were present in the frozen plasma and ST whole blood specimens in these two patients, clinical significance would not be expected. Thus, in general, the resistance profile analyses indicate that ST plasma and ST whole blood specimens gave similar drug resistance profiles when compared to frozen plasma specimens. SampleTanker specimens with discordant drug resistance profiles as compared to frozen plasma are summarized in Table 2.

Table 2.

Discordant Drug Resistance Profiles of Matched Frozen Plasma and ST Plasma and ST Whole Blood

		Frozen plasma				ST plasma and whole blood stored at ambient temperature for 2 and 4 weeks
Sample IDs	HIV-1 VL (log₁₀ copies/ml)	PI minor	PI major	NRTIs	NNRTIs	PI minor	PI major	NRTIs	NNRTIs
Discordant specimens for ST plasma
HN047	3.24			M184V	L100IL, K103N, P225H			L74LV, M184MV	L100IL, K103KN,
HN032	3.38			D67N, V75I, F77L, Y115F, F116Y, Q151M, M184V, K219E	L100I, K103N			D67N, V75I, F77L, Y115F, F116Y, Q151M, M184V, K219E	L100I, K103N, V179IT
HN117	3.50			M184V	G190S			L74V, M184V	G190S
HN100	3.87			T69N, M184V, K219EK	K103N, Y181C, H221HY			T69N, K70R, M184V, K219EK	K103N, Y181C, H221HY
Discordant specimens for ST whole blood
HN090	3.21								V90IV
HN035	3.26			V118I	K103R			V118I	K103R, V179DINV
HN032	3.38			D67N, V75I, F77L, Y115F, F116Y, Q151M, M184V, K219E	L100I, K103N			D67N, V75I, F77L, Y115F, F116Y, Q151M, M184V, K219E	L100I, K103N, V179IT
HN029	3.42							T69A
HN117^a	3.50			V118I, M184V	G190S			V118I
HN103^a	3.54			M184V	K103N, K238KN			M184V	K103N

These specimens had identical drug resistance mutation patterns at 2 and 4 weeks.

ST plasma and ST whole blood were stored at ambient temperature for 2 and 4 weeks.

VL, viral load; PI, protease inhibitor; NRTI, nucleoside reverse transcriptase inhibitor; NNRTI, nonnucleoside reverse transcriptase inhibitor.

Discussion

In the current study, we sought to expand dried blood specimen collection devices beyond filter papers by evaluating a non-paper-based matrix dried blood specimen collection and transportation device, SampleTanker^®. This device has a unique design that holds a relatively large volume of blood compared to DBS (1,000 μl vs. 50–100 μl), and it has a self-contained humidity indicator capsule for storage and transportation. This design limits the need for packaging materials and humidity monitoring device as in DBS packaging, storage, and transportation. Our results indicate that ST plasma (36.0% at both 2 and 4 weeks storage duration) had significantly reduced genotyping efficiencies when compared to frozen plasma specimens (Table 1). These results are in contrast to the earlier studies conducted by the manufacturers.^14

–20 In one of the studies,¹⁴ 20 dried ST plasma specimens made from HIV-1-positive archived plasma specimens with VLs ranging from 3.49 to 5.41 log₁₀ copies/ml and stored at ambient temperature for 4 to 7 days were genotyped using the TruGene HIV-1 genotyping assay. The authors reported 100% genotyping efficiency for both frozen plasma and dried ST plasma specimens. In another report, when frozen plasma VL levels were stratified into 3–4, 4–5, 5–6, and >6 log copies/ml, 55.6%, 78.4%, 95.2%, and 100% of the matched ST plasma specimens that had been exposed to ambient temperature for a mean of 19 days were successfully genotyped comparing to frozen plasma specimens using the standard TruGene HIV-1 Genotyping kit protocol.¹⁸ Thus the different genotyping efficiency rates could not be explained solely by the different study designs since the ST plasma specimens used in the 2005 study were exposed to ambient temperature during shipments from Israel to Atlanta, GA, and the mean shipment duration was 19 days, which falls within the ambient temperature exposure duration of our ST plasma specimens during storage. In addition, the broadly sensitive in-house genotyping assay used for the current study has been extensively validated with a genotyping sensitivity of ≥95% when the plasma VL is ≥3.00 log₁₀ copies/ml following our standard protocol of using 200 μl of plasma for extraction with the NucliSENS EasyMAG automated extraction system.^3,10,22,23 Thus, future studies may be needed to delineate the contradictory results obtained by different investigators.

However, it is somewhat surprising to notice that genotyping efficiencies of the ST whole blood specimens (49.0% and 42.9% when stored for 2 and 4 weeks, respectively) are not much better than the ones obtained with ST plasma specimens since previous studies of storage and transportation conditions using DBS specimens on the impact of genotyping efficiencies showed that DBS specimens were more stable and had higher genotyping efficiency rates than dried plasma/serum spots when they had been exposed to ambient temperature conditions for the same period of time.^10,28,29 Furthermore, our own recent study with DBS specimens stored at ambient temperature for 2 weeks and shipped at ambient temperature has revealed the storage and shipping conditions tested in the study had no effect on genotyping efficiency when compared with matched frozen plasma specimens collected from ART-failure patients using the broadly sensitive genotyping assay with the initial whole blood input of 100 μl for one DBS spot. The lower than expected genotyping rate (60–75%) with dried ST whole blood obtained by a previous study conducted by the manufacturer might be the results of the lower genotyping sensitivity related to using the TruGene HIV-1 Genotyping kit.^16,30 Due to the absence of independent studies beyond ones conducted by the manufacturer¹⁶ using ST whole blood specimens for HIVDR genotyping we believe that the jury is still out and further studies with larger sample sizes including VL measurement for ST specimens are needed to confirm our findings. We also want to point out the limitations of the current study. SampleTanker^® was not originally designed for the collection and transportation of dried whole blood specimens. Due to the surface tension of the matrix device and the viscosity of the whole blood during the specimen reconstitution process, we encountered difficulties for a few specimens to be fully reconstituted with elution buffer, which might result in some loss of recovered whole blood specimens leading to lower genotyping efficiencies. In addition, the relatively large volume of specimens (1 ml) that STs could hold might also be a negative factor in term of preserving the integrity of the dried blood specimens as indicated in the previous study¹⁴ as well as our study since we had to dry the STs at ambient temperature for at least 16 h in order to ensure the complete dryness of the STs (data not shown).

Despite the significantly lower genotyping efficiencies seen in the current study for both ST plasma and ST whole blood specimens than in frozen plasma specimens, the overall resistance profile concordance rate was high (97%), which is in agreement with previous findings^14

–20; with the exception for one patient, all detected discordant mutations either had no clinical significance, or the discordant mutations were compensated by other DRMs found in the same patients. These kind of compensations for missing DRMs by other DRMs in a particular patient would always be present, especially in heavily ART-treated patients as in this cohort (median time on ART=2.5 years). The DRMs found in the ST whole blood specimens, not in the frozen plasma, might come from archived viruses that had integrated into the cellular genome. Most of the DRMs detected were secondary mutations and/or polymorphic sites that would not induce resistance or would induce resistance only when combined with other mutations.^24
–26 We believe that if the design and reconstitution process can be improved for dried ST whole blood specimens and further studies can be conducted with the newly improved STs with a broad VL range to confirm the comparable genotyping efficiency to frozen plasma, SampleTanker^® may still hold a future for HIVDR monitoring surveys in resource-limited settings.

In conclusion, we have performed an evaluation of STs for HIVDR genotyping in a resource-limited country under field conditions, and we have demonstrated that with the current design, the device may not be suitable for dried blood specimen collection, storage, and transportation for HIVDR genotyping. Giving the simplicity of ST specimen collection, packaging, storage, and transportation, the relatively large volume sampling capacity compared to DBS, and the high agreement of resistance profiles between ST whole blood and frozen plasma specimens, more studies using an improved design of the ST may warrant further evaluation of its genotyping efficiency for the monitoring of ART patients in resource-limited settings.

Footnotes

Acknowledgments

Dr. Guoqing Zhang was a recipient of the 2010–2011 International Emerging Infectious Fellowship (IEID) sponsored by the American Public Health Laboratory (APHL) and CDC. This research has been supported by the President's Emergency Plan for AIDS Relief (PEPFAR) through the Centers for Disease Control and Prevention.

Use of trade names is for identification only and does not constitute endorsement by the U.S. Department of Health and Human Services, the Public Health Service, or the Centers for Disease Control and Prevention. The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention/the Agency for Toxic Substances and Disease Registry.

Author Disclosure Statement

No competing financial interests exist.

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