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Published in final edited form as: J Clin Virol. 2020 Nov 25;135:104694. doi: 10.1016/j.jcv.2020.104694

Field Evaluation of HIV-1 Viral Load Monitoring in Adults and Children Receiving Antiretroviral Treatment in Nigeria by Dried Blood Spot Testing with RealTime HIV-1 on m2000

Monday Tola a, Habib O Ramadhani b, Sylvia Adebajo c, Trevor A Crowell d,e, Rebecca G Nowak b, Manhattan E Charurat a,b, Patrick Dakum a,b, Nicaise Ndembi a,b,f,g,#
PMCID: PMC9518813  NIHMSID: NIHMS1795039  PMID: 33476928

Abstract

In resource-limited settings, use of dried blood spots (DBS) could be a pragmatic alternative to plasma for VL monitoring in people living with HIV (PLWH). We compared results from DBS to standard plasma VL testing under field conditions in patients receiving antiretroviral therapy (ART). DBS cards were prepared from venous blood (V-DBS), finger-pricks using micro-capillary tubes (M-DBS), and direct spotting (D-DBS). DBS and matched EDTA plasma were tested on the Abbott m2000 platform using the appropriate RealTime HIV-1 quantitative CE protocol. Matched plasma samples were also tested on the Roche COBAS Ampliprep/COBAS TaqMan version 2.0. Diagnostic accuracy indicators (sensitivity, specificity, misclassification rate, and kappa coefficient) for viral failure (VF) based on different VL threshold levels and agreement of absolute VL were calculated. A total of 669 participants provided 2,676 samples. V-DBS had a peak sensitivity for VF of 89.1% [95% CI: 85.5–92.7] at the 1000 copies/mL threshold and a peak specificity of 97.4% [95% CI: 95.9–99.0] at the 5000 copies/mL threshold. The lowest proportion of upward misclassification (patients classified with VF who actually had viral suppression) for V-DBS was 3.1% [95% CI: 1.4–4.8] at the 5000 copies/mL threshold, whereas the lowest proportion of downward misclassification (patients classified as undetectable who actually had VF) was 10.9% [95% CI: 7.2–14.5] at the 1000 copies/mL threshold. Abbott RealTime HIV-1 VL results from all 3 DBS types for adults and children showed strong correlation with the gold standard plasma-based assay. DBS could be useful for monitoring VL in resource limited settings such as Nigeria.

INTRODUCTION

According to UNAIDS and the National Agency for the Control of AIDS (NACA), Nigeria has HIV prevalence of 1.4%, representing approximately 1.9 million people living with HIV (PLWH) in the country [1]. Immunologic and clinical markers for treatment monitoring used to be recommended in resource-limited settings (RLS) [2], but these markers have led to misclassification of treatment failure and accumulation of drug resistance mutations that may compromise future treatment options [3]–[6]. As a result of this, recent World Health Organisation (WHO) guidelines recommend routine viral load (VL) monitoring of patients on antiretroviral therapy (ART) to identify viral failure (VF) [7]. This strategy reduces morbidity and mortality because of the prompt detection of treatment failure and early switch to effective alternative therapy [8]. Routine VL monitoring is also emphasized as part of the UNAIDS 95–95-95 initiative that provides global targets to diagnose 95% of PLWH, get 95% of PLWH on ART, and ensure that 95% of PLWH on ART achieve VL suppression [9]. However, obstacles to patients’ VL monitoring in RLS include the costs associated with running supportive laboratories, inefficient specimen transport systems, and challenges with the implementation of laboratory-related quality assurance measures [10].

Dried blood spots (DBS) are included in recent WHO guidelines for specimen collection [7] and may prove to be a useful and efficient tool for VL monitoring [11]–[13]. Advantages of DBS over the traditional plasma specimen collection include ease of collection, transport and storage. DBS can be kept at ambient temperature with high humidity without degradation of the viral nucleic acids for at least 2 weeks before samples should be refrigerated or frozen [14], [15]. Several studies have demonstrated that this type of sample may be used for monitoring of HIV drug resistance [11], [16], [17]. The main disadvantages of using DBS for VL testing include the time and effort required for cutting of cards, sterilization of forceps, and incubation before performing the assay, as well as decreased sensitivity to detect low levels of viremia when compared to plasma-based testing[13], [15].

The Abbott RealTime HIV-1 assay sample preparation (mSystem RNA sample preparation) selectively captures RNA, resulting in assay specificity of ≥90% when using the 1000 copies/mL VF threshold for plasma samples [18]. A number of studies conducted using the initial DBS protocols developed to guide its use for VL monitoring, demonstrated a sensitivity and specificity of 95.2% and 91,7%, of this method respectively [19]. We evaluated the accuracy of the CE-marked one-spot DBS HIV-1 VL protocol introduced by the manufacturer in 2016 against plasma VL testing under field conditions, and compared the data collected from adults and children in Nigeria using different DBS collection methods, to the plasma sample type.

MATERIALS AND METHODS

The study participants were PLWH on ART accessing care in four Nigerian states with high HIV prevalence namely: Kano, Katsina, Nasarawa, and the Federal Capital Territory. Sites with a high volume of plasma VL testing (100 or more VL tests/month) and the use of DBS for early infant diagnosis were selected for study participants’ recruitment. Study participants were PLWH who had accessed routine clinical care from September 2017 to October 2018 at the study sites, and who had been prescribed ART for at least six months prior to the commencement of the study. Only participants who provided written informed consent (and assent by children 12 – 17-years-old in addition to parent/guardian consent) were enrolled in the study.

Random anonymous identification numbers were assigned to each study participant. Venous and capillary blood samples were collected from each participant and pair plasma and DBS specimens prepared for each participant from the sample. Demographic information and ART prescription history were extracted for each participant from routine VL request forms.

Plasma specimens were prepared from the venous blood drawn into an EDTA collection tube within 3 hours of collection by centrifugation at 1.600xg for 10 minutes. DBS were prepared from venous blood (V-DBS) by spotting 70 µL of blood from an EDTA tube onto each of the 5 preprinted circles on a Munktell TFN card (Ahlstrom Germany GmbH) using a calibrated micropipette with disposable aerosol barrier pipette tips. Two additional DBS cards were prepared from finger-prick capillary blood by spotting 3 drops of blood from a 70 µL microcapillary tube onto each preprinted circle on a card (M-DBS) and by directly spotting 3 drops of blood from the finger-prick site onto each circle on a card (D-DBS) using the same brand of Munktell TFN card (Ahlstrom Germany GmbH). Overall, there were 3 cards (of different collection methods) with 5 replicate circles for each patient. Prepared DBS cards were dried at ambient temperature overnight on a drying rack, placed individually in a glassine envelope within a zip-lock bag containing 5 desiccant packs and 1 humidity monitor. All samples were transported to the Reference Laboratory at the Institute of Human Virology Nigeria within a week of sample collection. Plasma samples were shipped on dry ice and DBS cards were shipped at ambient temperature.

VL measurements were independently performed using plasma and DBS samples. All samples were tested using the Abbott m2000sp (sample preparation)/Abbott m2000rt (real-time) system (Abbott Laboratories, Germany). The RealTime HIV-1 0.6 mL sample input protocol was used for plasma analysis [20]–[22], while DBS cards were processed utilizing the RealTime HIV-1 Quantitative DBS version 4.0 protocol [22] which uses one completely filled pre-perforated spot for assay per sample type. Both sample types were processed as per manufactureŕs recommendations [21]. Quantitative assay results were reported as copies/mL or log copies/mL [20].

Statistical Analyses:

The median and interquartile ranges (IQR) of the VL for all the samples and the samples by type of collection, were computed. The agreement of absolute VL results between V-, M-, and D-DBS and plasma was assessed using Bland–Altman plots. Diagnostic accuracy and were the optimal DBS VF threshold was determined by calculating the sensitivity, specificity, misclassification rate, and kappa coefficient of V-, M-, and D-DBS for VF (defined by plasma VL ≥1000 copies/mL). The analyses of VF were conducted for 3 DBS VF thresholds (1000, 3000, and 5000 copies/mL) to ascertain the effect on clinical misclassification rates. Downward misclassification (false-negative for diagnosis of VF) was defined as a sample ≥1000 copies/mL by plasma, but <1000, 3000, or 5000 by DBS for each respective DBS-threshold analysis. Upward misclassification (false-positive for diagnosis of VF) was defined as a sample <1000 copies/mL by plasma, but ≥1000, 3000, or 5000 by DBS for each respective DBS-threshold analysis. These analyses were performed for the study population, and separately for children and adults.

As a quality check, plasma results from the Roche COBAS Ampliprep/COBAS TaqMan (CAP/CTM) HIV-1 v2.0 assay (a platform routinely used in the implementation of the PEPFAR program in Nigeria) were compared with the Abbott RealTime HIV-1 assay. Any participant with greater than 0.7 log copies/mL difference between the 2 plasma results was removed from the final data set, as per standard quality assurance practice [23]. Differences between DBS performance of adult and children samples at different testing platforms, DBS types, and VL thresholds were compared by examining the corresponding 95% confidence interval (CI). Statistical analyses were performed using SAS version 9.3 (SAS Institute Inc., Cary, NC).

RESULTS

A total of 2,676 samples were collected from 669 PLWH - 490 adults with median age of 37 years (IQR 32 – 43) and 179 children with median age of 10 years (IQR 7 – 12). Also, 72% of the adult participants and 58% of the children participants were female. In addition, 53% of children had a VL above 1000 copies/ml [with median VL (IQR) of 3.32 log copies/ml (1.59 – 4.67)] while 38% of adults had VL above 1000 copies/ml [with median VL (IQR) of 1.59 log copies/ml (1.59 – 4.12)] (Table 1).

Table 1.

Demographics and Clinical Characteristics of Persons Living with HIV Prescribed Antiretroviral Therapy at Four Nigerian Clinics

Characteristics Children (n=179) Adults (n=490)
Median age (IQR) 10 (7 – 12) 37 (32 – 43)
Total patients (Median females) 104 (58.10) 353 (72.04)
VF, n/N (Median)
VF overall 98/179 (54.75) 186/490 (37.96)
VF among females 53/104 (50.96) 133/353 (37.68)
VF among males 45/75 (60.0) 53/137 (38.69)
Median VL (IQR)
VF overall 3.32 (1.59 – 4.67) 1.59 (1.59 – 4.12)
VF among females 3.16 (1.59 – 4.67) 1.59 (1.59 – 4.14)
VF among males 3.68 (1.59 – 4.68) 1.62 (1.59 – 3.93)
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Abbreviations: IQR, Interquartile range; VF, Virologic failure = Viral Load >1000; n, number of participants who failed; N, Total number of participants.

Table 2 shows the sensitivity, specificity and kappa agreement with misclassification rates at VL thresholds of 1000, 3000 and 5000 copies/ml. As the DBS VL threshold increased, the sensitivity for predicting VF decreased and specificity increased. Also, as the VL threshold increased, the proportion of upward misclassification decreased while the proportion of downward misclassification increased. The lowest proportion of upward misclassification (patients classified with VF who actually had viral suppression) for Abbott V-DBS was 3.1% [95% CI: 1.4 – 4.8] at 5000 copies/mL threshold, while the lowest proportion of downward misclassification (patients classified as undetectable who actually had VF) was 10.9% [95% CI: 7.2 – 14.5] at the 1000 copies/mL VL threshold. No significant difference was observed in the sensitivity, specificity, misclassification and agreement between DBS types within each VL threshold.

Table 2:

Sensitivity, Specificity, Misclassifications and Kappa Agreement Using DBS Testing Platform (Abbott), DBS type and VL Threshold on Patients Receiving Antiretroviral Therapy Compared with Plasma Among Adults and Children (Nigeria, 2017 – 2018)

VL Threshold Plasma: DBS Sample Type (n = 669) Sensitivity % (95% CI) Specificity % (95% CI) Upward Misclassification % (95% CI) Downward Missclassification % (95% CI) Kappa Agreement % (95% CI)
1000:1000 Abbott M-DBS 85.2 (81.1 – 89.3) 91.5 (88.7 – 94.3) 8.5 (5.7 – 11.3) 14.8 (10.7 – 18.9) 77.0 (72.1 – 81.9)
Abbott D-DBS 85.6 (81.5 – 89.6) 90.9 (88.1 – 93.8) 9.1 (6.2 – 11.9) 14.4 (10.3 – 18.5) 76.8 (71.8 – 81.7)
Abbott V-DBS 89.1 (85.5 – 92.7) 86.6 (83.2 – 90.0) 13.4 (10.0 – 16.8) 10.9 (7.2 – 14.5) 74.9 (69.9 – 80.0)
CAP/CTM V-DBS 85.2 (81.1 – 89.3) 97.9 (96.5 – 99.4) 2.1 (0.7 – 3.5) 14.8 (10.7 – 18.9) 84.5 (80.4 – 88.6)
1000:3000 Abbott M-DBS 72.5 (67.3 – 77.7) 96.9 (95.2 – 98.6) 3.1 (1.4 – 4.8) 27.5 (22.3 – 32.7) 71.7 (66.4 – 77.0)
Abbott D-DBS 71.5 (66.2 – 76.7) 96.6 (94.9 – 98.4) 3.4 (1.6 – 5.1) 28.5 (23.3 – 33.8) 70.4 (65.0 – 75.8)
Abbott V-DBS 76.4 (71.5 – 81.3) 96.1 (94.2 – 98.1) 3.9 (1.9 – 5.8) 23.6 (18.6 – 28.5) 74.4 (69.2 – 79.5)
CAP/CTM V-DBS 79.6 (74.9 – 84.3) 100.0 (100.0 – 100.0) 0.0 (0.00 – 0.00) 20.4 (15.7 – 25.1) 81.8 (77.4 – 86.2)
1000:5000 Abbott M-DBS 65.8 (60.3 – 71.4) 97.2 (95.5 – 98.8) 2.8 (1.2 – 4.5) 34.1 (28.6 – 39.7) 65.6 (60.0 – 71.4)
Abbott D-DBS 62.7 (57.1 – 68.3) 97.4 (95.9 – 99.0) 2.6 (1.0 – 4.1) 37.3 (31.7 – 42.9) 62.9 (57.1 – 68.9)
Abbott V-DBS 67.2 (61.8 – 72.7) 96.6 (95.2 – 98.6) 3.1 (1.4 – 4.8) 32.7 (27.3 – 38.2) 66.7 (61.1 – 72.3)
CAP/CTM V-DBS 75.0 (70.0 – 80.0) 100.0 (100.0 – 100.0) 0.0 (0.00 – 0.00) 25.0 (20.0 – 30.0) 77.6 (72.8 – 82.4)
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Abbreviations: M-DBS, Micro-capillary Dried Blood Spot; D-DBS, Direct Dried Blood Spot; V-DBS, Venous Dried Blood Spot; VF, Virologic failure; n, number of participants who failed

Figure 1 shows the correlation between VL measured on plasma using the Roche platform commonly used in Nigeria and the Abbott. The correlation was 0.64 between V-DBS and Abbott plasma, 0.65 between M-DBS and Abbott plasma, and 0.67 between D-DBS and Abbott plasma (Figure 1B–D). All the correlations were moderate and statistically significant.

Figure 1.

Figure 1.

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Correlation between viral load (VL) measurements on plasma and on dried blood spots (DBS).

Figures 2A–D shows the Bland-Altman analysis of the VL measured on plasma using the Roche platform commonly used in Nigeria and the Abbott. A strong correlation was observed (Figure 2A), with mean difference (bias) of −0.27 log copies/ml. There was a bias of −0.03 log copies/ml between M-DBS and Abbott plasma, −0.04 log copies/ml between D-DBS and Abbott plasma, and −0.07 log copies/ml between V-DBS and Abbott plasma (Figures 2B–D).

Figure 2.

Figure 2.

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Bland–Altman mean difference analysis between Abbott plasma and Abbott V-, M-, and D-DBS and CAP/CTM V-DBS

DISCUSSION

The two main platforms used for HIV-1 VL testing in Nigeria (CAP/CTM and Abbott m2000) have shown good sensitivity for DBS VL, which is similar to the results observed in other studies [24], [25], while the specificity was relatively higher for the Abbott assay. This difference can be attributed to the capacity of the Abbott HIV-1 VL assay to exclude contamination from HIV-1 pro-viral DNA and viral-RNA from peripheral blood mononuclear cells in RT-PCR [12], which is due to its RNA selective extraction technology [22], while CAP/CTM extraction using silica beads and total nucleic acid extraction cannot differentiate between HIV-1 DNA and RNA [26]. This low specificity can lead to unnecessary repeat VL testing and potentially unnecessary switch to second and third-line ART regimens.

VL testing by DBS has many advantages over plasma VL and is recommended for resource-limited settings that are scaling up routine VL testing in PLWH [27]. Several studies have presented good concordance of plasma and V-DBS [28], [29], M-DBS [28],[19] and D-DBS [30] using different platforms. However, this study used a combination of all 3 methods of sample collection preparing DBS from the same patients. Comparison of VL results from all DBS sample types (M-DBS, D-DBS and V-DBS) obtained from the Abbott platform revealed strong correlation with the gold standard of Abbott plasma, showing that DBS options are acceptable substitutes to plasma VL in the identification of VF patients. DBS may therefore be a useful tool for deploying VL testing in resource-limited settings that lack access to traditional plasma-based assays, including rural areas. Our findings were similar to other studies conducted using the Abbott m2000 platform in Malawi, Kenya, and Spain [19], [25], [31]. In our study, sensitivity and specificity to detect treatment failure at 3 different thresholds were comparable across the 3 sample types and similar to previously published studies [12], [31].

Identifying an appropriate sample type and threshold for VF are critical, since decisions regarding ART management are taken primarily from VL measurements above or below the defined threshold for failure. A review of the use of DBS for VL monitoring showed that DBS VL results may vary based on factors such as punch size, punch method, adequacy of volume collected, elution buffer used, and sample volume after extraction [32].

The high VL observed in this study was expected being a targeted VL and invited patients with initially high VL result (within 6 months before initiation of the study according to the national algorithm). This is supported by the 2016 WHO consolidated guidelines which recommend routine VL testing as the preferred method for monitoring patients on ART and modified the VF threshold for DBS to 1000 copies/mL [33]. Two key parameters to look at for every platform used for VL are the upward and downward misclassification. We found that few virally-suppressed PLWH would be misclassified as failing ART and this number decreased further as the VL threshold increases. These low values are very important for any platform and help eliminate unnecessary ART switches, though WHO guidelines suggest a repeat VL would be performed before any treatment switch, increasing the likelihood for subsequent correct classification and avoiding an unnecessary regimen change.

Considering the definition of downward misclassification, minimizing this parameter is a necessity for clinical and programmatic purposes. This study showed that the most favorable threshold using the Abbott platform, for the determination of VF using DBS is the 1000 copies/ml. At this threshold, false negative misclassification is lowest for the 3 DBS sample types with no significant difference between the sample types. The V-DBS had a lower value (10.9, 95% Cl: 7.2 – 14.5) compared to that of CAP/CTM (14.8, 95% Cl: 10.7 – 18.9). According to the WHO guidelines, these patients would not be scheduled for a repeat VL until 12 months later or until they develop signs of treatment failure and thus would be continued on a failing ART regimen. If this parameter is high, there will be an increased risk of clinical and immunologic failure, development of HIV drug resistance mutations, and transmission of HIV [34]. Our study had a number of strengths and limitations. All evaluations were conducted in the same laboratory, on the same patient samples, and by the same three technical staff with several years of experience with VL testing. However, staff were not blinded to the results of the reference test. This study was conducted with field collected samples in routine practice and in a laboratory not dedicated to research, which is simple and practical for resource-limited settings. Lower level technicians and health care workers in remote sites can be supported to make valid DBS samples for VL monitoring, as demonstrated by prior proficiency with DBS for early infant diagnosis. The M-DBS and D-DBS methods are of particular interest in pediatric populations in whom venipuncture could be challenging.

It is now accepted that the use of DBS is the most feasible way to provide VL monitoring to patients especially in hard to reach areas of resource-limited settings [35], [36]. In Nigeria, this could include highly-marginalized populations of men who have sex with men and transgender women that are disproportionately impacted by HIV and face unique barriers to accessing the limited available resources for HIV care [37],[38],[39]. Furthermore, DBS can also allow monitoring of HIV drug resistance in patients with VF [11], [12], [16].

CONCLUSIONS

Good correlation and agreement were observed between matched DBS and plasma samples for the Abbott platform. Our findings showed that DBS may be a reliable alternative specimen to plasma for HIV-1 VL monitoring using the Abbott platform. Policy makers and HIV program implementers can decide which method(s) of the DBS preparation are the most suitable depending on the local context and availability of resources to sustain supplies, manage biological waste and availability of human capacity. This finding contributes to the growing body of evidence supporting the use of DBS for VL testing, as current guidelines state that clinical management regarding ART maintenance or switch be based on VL >1000 copies/ml. In this study, the measurement of HIV-1 VL from DBS with the Abbott RealTime HIV-1 assay on the m2000 system performed well on all three DBS sample types tested.

Acknowledgements:

This program evaluation has been supported by the President’s Emergency Plan for AIDS Relief (PEPFAR) through the Centers for Disease Control and Prevention (CDC) under the terms of U2G GH002099-01, PA GH17-1753 (ACHIEVE), a grant from Abbott Molecular Diagnostics, Roche Molecular Systems, and the National Institute of Health/National Institute of Allergy and Infectious Disease (R01 AI147331-01).

We acknowledge Dr. Swaminathan Mahesh of the US Centre of Disease Control and Prevention and Prof. Morenike Oluwatoyin Folayan, Department of Child Dental Health, Obafemi Awolowo University, Ile-Ife, Nigeria for carefully reviewing this manuscript.

Funding:

This work was supported by the US CDC PEPFAR U2G GH002099-01, PA GH17-1753 (ACHIEVE) and US NIH R01 AI147331-01.

Footnotes

Portions of these data were presented as poster #432 at the International AIDS Society (IAS) Conference on HIV Science, 21–24 July 2019, Mexico City, Mexico.

Ethics statement

This study was reviewed and approved by the National Health Research and Ethics Committee of Nigeria (NHREC Approval # NHREC/01/01/2007 – 10/12/2018B) and the University of Maryland Baltimore Institutional Review Board (UMB CICERO #HP00066914).

Availability of data and material: The data that support the findings of this study are available from the corresponding author [NN], upon reasonable request.

Conflicts of interest: The authors have no conflicts of interest.

Publisher's Disclaimer: Disclaimer: This work was primarily funded by PEPFAR. The funding source had no role in the study design, data collection, data analysis, data interpretation, or writing of the report. The authors had full access to all the data related to this analysis and independently made the decision to submit the findings for publication. The findings and conclusions in this publication are those of the author(s) and do not necessarily represent the official position of the Centers for Disease Control and Prevention (CDC), the U.S. Army, Department of Defense or the Department of Health and Human Services. The investigators have adhered to the policies for protection of human subjects as prescribed in AR-70.

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