Introduction
Sub-Saharan Africa remains the worst hit by the HIV/AIDS epidemic with 20.7 million of the global estimated 38 million HIV-infected individuals by 2020 [
1]. A tremendous achievement has been realised in increasing access to antiretroviral therapy (ART) among individuals living with HIV [
1]. This is a huge milestone in combating the epidemic since ART suppresses viral replication, reduces the morbidity associated with HIV/AIDS and curbs HIV transmission to uninfected individuals [
2]. Increased access to ART comes with a challenge of increasing prevalence of HIV drug resistance (HIVDR) [
3,
4] which compromises ART benefits [
5]. Monitoring of HIVDR remains a priority in the WHO strategy for combating the HIV/AIDS epidemic. The new UNAIDS 2025 targets call for 95% of HIV-infected individuals becoming aware of their status, initiation of 95% of those with known HIV-positive status on treatment and having 95% of those on ART achieving virological suppression [
6,
7]. To achieve these targets, virological monitoring and HIV genotypic resistance testing of individuals on ART are necessary as recommended by the WHO [
7]. Viral load testing provides a basis for ART response monitoring and guides the need to switch drug regimen classes [
2]. Guidelines in resource-rich settings recommend HIV-infected individuals to undergo viral load testing at ART initiation or modification. Due to transmitted HIVDR, genotypic resistance testing for mutations in the reverse transcriptase and protease genes is recommended prior to ART initiation or modification to guide selection of ART regimen [
8]. In contrast, guidelines in resource-constrained settings like Uganda recommend viral load testing for adults 6 months post -ART initiation and subsequently every 12 months for those with viral load suppression. Also, genotypic resistance testing is only recommended for adult individuals failing on 2nd line and 3rd line regimes, and those failing on protease inhibitor-based or dolutegravir-containing regimens [
2]. To circumvent economic barriers that impede ART monitoring in resource-constrained settings, the WHO has recommended a relatively cost-effective public health approach for surveillance of acquired [
9] and pre-treatment [
10] HIVDR in resource limited settings. However, this too, requires HIVDR genotyping.
HIV-1 genotypic testing is a nucleic acid amplification and sequencing test aimed at detecting the existence of HIVDR mutations in targeted regions of the HIV-genome such as protease, reverse transcriptase and integrase genes [
11,
12]. It also examines HIV diversity and generates data used for molecular epidemiological and evolutionary studies [
13,
14]. The interpretation of HIVDR genotyping data is done by a clinical expert or with the aid of an HIVDR database [
15]. HIV genotyping consists of, extraction and purification of the viral nucleic acid, amplification of the target gene and sequencing of the amplicons.
Plasma specimens are preferred to dried plasma/serum and blood spot specimens for HIV genotyping in most research studies and clinical applications [
16]. This is because plasma sequence data represents actively-replicating viruses which form a large proportion of viruses circulating in the body [
17]. However, the need for cold-chain and associated transportation and storage costs have made plasma less appropriate in resource-constrained settings, especially rural areas. Consequently, dried blood spot (DBS) specimens offer a cheaper alternative to the conventional use of plasma for virological monitoring [
18‐
20] and HIV genotypic resistance testing [
21‐
25]. The preference of DBS to plasma specimens is because DBS specimens are easy to collect, process, transport and can be kept at ambient temperature for relatively longer time and thus do not require a costly and a not-readily available cold-chain in resource-constrained settings [
18,
21,
22]. Furthermore, many studies report concordance between paired plasma and DBS specimens for HIV genotyping [
21‐
23,
25,
26], although one study reported discordance based on next-generation sequencing [
27]. Genotyping success rates of DBS specimens are, however, continually variable due to several factors.
Following the renewed global efforts to eliminate the public threat posed by HIV/AIDS [
6], HIV genotypic drug resistance testing is crucial to the success of ART programmes. The clinical community is reliant on results of HIV genotypic tests in making important clinical decisions such as which drug regimen to offer and when to switch to another regimen class. It is therefore imperative for laboratories to ascertain the success rates of specimens they analyse. Currently in our setting, there is a paucity of data on the genotyping success rates of unpaired DBS and plasma specimens. In the current study, we retrieved the data of DBS and plasma specimens analysed in the MRC/UVRI and LSHTM virology laboratory between 2016 and 2019 and evaluated the genotyping success rates of both DBS and plasma specimens. In addition, we assessed the possible correlates of genotyping failure of both specimen types. This data is vital for quality assurance assessment and is informative in decisions for improving not just the performance of the laboratory assays but also the management of studies and clinical facilities from which specimens are obtained.
Discussion
HIV drug resistance genotyping has revolutionised the clinical management of individuals on ART because it informs decisions like when to implement regimen switch. Successful genotyping in the laboratory is critical to patient care. In this cross-sectional study, we examined the genotyping success rates of unpaired DBS and plasma specimens analysed in a WHO-designated HIVDR laboratory in a resource-constrained setting in Uganda and assessed the correlates of specimen genotyping failure.
From 2016 to 2019, we obtained an overall GSR of 65.7% for all specimens delivered for analysis in our laboratory, but the overall GSR for plasma was higher (85.9%) than that of DBS specimens (49.8%) over that 4-year period. The plasma genotyping success rate in this study was lower than the 100% genotyping success rate that was realised in our previous study [
37]. Similarly, the genotyping success rate of DBS specimens reported here is also lower than that reported by Zhang et al. in a survey in Kenya and Tanzania [
38]. The comparatively higher genotyping success rates in those studies could be attributed to them being well controlled research studies in which the study staff were well trained, monitored, and specimen collection, transportation and storage done according to well-designed protocols. In contrast, this study had specimens from both research studies and routine clinical diagnosis including cross-border specimens that required lengthy shipping procedures, with most specimens from routine clinical diagnosis contributing significantly to genotyping failure (p < 0.0001). Though not part of our analyses, we have in some cases received DBS specimens 3 months from the collection date. Aware of inadequate facilities in most of these facilities, it is possible that DBS specimens shipped to our laboratory for genotyping were probably not handled under optimum conditions that maintain sample integrity.
Studies have shown that better genotyping success rates are realised when DBS specimens are kept at ambient temperatures for 2 weeks and stored frozen at – 80 °C prior to shipping, and that prolonged storage for over 2 weeks at ambient temperature reduced genotyping success rates [
22,
39]. Furthermore, most research studies prepared their DBS cards with venous blood while most routine clinical facilities preferred finger prick DBS. Parry et al. showed that venous DBS had better genotyping success rates compared to finger prick DBS [
22]. This probably explains why specimens from research studies had better genotyping success rates. It is probable that the site of collection of blood for DBS specimen preparation could have impacted on the genotyping success rates of the DBS samples. It will be interesting for future studies to assess the various DBS preparation methods and their impact on GSR of DBS specimens.
Generally, the genotyping success rates of DBS specimens were substantially lower than those of plasma over the 4 years. Our analysis revealed that over the 4 years, the overall GSR rapidly decreased with increasing number of DBS specimens presented for genotyping. The observation that the overall GSRs were decreasing in a similar trend as genotyping success rates of DBS specimens, further asserts that DBS specimens, to a large extent, contributed to overall genotyping failure. Our logistic regression analysis showed that DBS were at 90% more odds of genotyping failure compared to plasma specimens (aOR 0.10; 95% CI: 0.08–0.14; p < 0.0001) correlating DBS specimens to genotyping failure. This probably arose from the large proportion of DBS specimens originating from routine clinical diagnosis that face significant challenges in terms of specimen collection, transportation, and storage. Moreover, DBS have considerably gained preference to plasma because DBS specimens are easy to collect, transport and do not require a cold chain [
22,
40]. This explains why we received more DBS than plasma specimens between 2017 and 2019. The increase in DBS shipped to our laboratory was also attributed to the roll out of DBS for the national viral load programme and the subsequent use of remnant DBS for HIVDR testing among patients failing ART in Uganda. This popularity should be followed by precautions to ensure that specimen quality is not compromised on the way to the laboratory for genotyping. Evidence from other studies suggests that DBS specimen yield appreciable genotyping rates when specimens are properly handled to ensure nucleic acid integrity [
38,
41].
The higher genotyping success rates of plasma compared to DBS specimens reported here mirrors with findings of other researchers [
26,
42,
43]. The better GSR of plasma could probably be attributed to a higher rate of HIV-1 RNA degradation for DBS compared to plasma because of the extra processing time at ambient temperature. Temperature and humidity at which DBS specimens are stored affects the stability and integrity of nucleic acids embedded on filter papers [
44]. Despite the lower GSR of DBS compared to plasma in our HIVDR genotyping laboratory, we cannot undermine the reliability of DBS specimens as a suitable alternative to plasma especially in resource-constrained settings. Studies by Rottinghaus et al. [
45] and Diouara et al. [
46] both corroborate the suitability of DBS for HIV drug resistance testing.
For both DBS and plasma specimens, we noted a significant association between VL levels and HIVDR genotyping rates: As expected, VLs < 10,000 copies/mL were associated with genotyping failure (p < 0.0001). A similar observation was also noted when DBS specimen were analysed alone. In studies that examined the use of DBS for genotyping, DBS with VL < 10,000 copies/mL had reduced genotyping success rates [
22,
27]. Our findings also concur with studies that cited VL as the major determinant of concordance between plasma and DBS especially when viral load ≥ 5000 copies/mL [
21,
25,
27,
47]. Studies analysing paired DBS and plasma specimens report significant correlation and strong concordance in RNA levels between plasma and DBS specimens [
48,
49]. Similar to findings in this study, viral loads considerably influenced genotyping success rates in a survey carried out in Kenya and Tanzania where DBS specimens with VLs ≥ 5000 copies/mL achieved genotyping success rate of 90% [
38]. Interestingly, in our study, a substantial proportion of specimens had VL < 10,000 copies/mL and could explain why the overall GSR was only 65.7%. This implies that in this era when virological suppression (VL < 1000 copies/mL) rates nearing or exceeding 90% have been observed [
37,
41], and following recommendations to use more potent dolutegravir-containing regimens, genotyping success rates are likely to continue to decline. There is evidence in this study that shows median viral loads of specimens analysed in our laboratory rapidly decreasing from 2016 to 2019. This can be explained by rapid scale-up of ART following recommendations of the WHO and subsequent high VL suppression rates reported in our setting [
41]. What is more intriguing is that even at VL < 1000 copies/mL, HIVDR variants can still be selected as reported in a Kenyan study in which plasma samples with VL ≤ 1000 copies/mL had a genotyping success rate of 32% [
50]. Ultimately, robust techniques need to be adopted to ensure that genotyping assays are adapted to successfully genotype both plasma and DBS specimens at VL < 1000 copies/mL. Some studies have already demonstrated the possibility of successful genotyping at low and undetectable viremia [
50‐
52].
The findings of our study also highlight the importance of virological monitoring. Our findings show that 53.9% of the specimens analysed were brought for genotyping without prior viral load testing. This is expected in our setting where viral load testing is not widely available for all HIV-infected individuals due to high costs. We noted significantly higher odds of genotyping failure in specimens in which prior viral load testing was not done, both in the general analysis of all samples (aOR 0.64 95% CI: 0.13–0.56; p = 0.001) and even when DBS specimens were analysed alone (OR 0.68 95% CI: 0.6–0.77; p < 0.0001). It is possible that the specimens that were not subjected to pre-genotyping VL testing had VL < 5000 copies/mL, the range at which genotyping failure is more likely [
22,
38]. Pre-genotyping viral load testing could be essential in qualifying specimens for HIVDR genotyping. Without pre-genotyping VL testing, time and resources may be wasted on genotyping non-eligible specimens resulting in low GSR and increasing the turn-around time. A study in Malawi reported that a delayed turnaround time in their laboratories was associated with lack of viral load testing and the use of DBS compared to plasma [
53]. In our unpublished data, we noted that paediatric plasma and DBS samples with substantial genotyping failure turned out to be virally suppressed when we did viral load testing.
Despite the challenges of using plasma specimens in our setting, they still yield better genotyping success rates than DBS. The use of DBS specimens is a plausible option to plasma and whole blood due to its advantages which include the ease of collection, transportation, and storage. The WHO recommends the use of DBS specimens for viral load monitoring in resource-constrained settings where infrastructural and logistic deficiencies hinder the use of plasma specimens. Interestingly, a viral load threshold of ≥ 1000 copies/mL used to define virological failure with plasma specimens is also applicable to DBS specimens [
7,
16]. Studies collaborate this with findings of high sensitivity and specificity of DBS specimens at a VL ≥ 1000 copies/mL [
32,
54]. Any form of specimen that is used in HIVDR genotyping requires maximum effort towards ensuring that specimen collection, processing, transportation, storage, and testing is done appropriately so that accurate and reliable results are obtained.
This cross-sectional study is limited by several factors: The specimens analysed came from multiple sources, some of which were routine clinical diagnosis laboratories, which are not research-focused, thus, strict adherence to SOPs for proper specimen collection and handling cannot be guaranteed. Therefore, the widespread genotyping failure in most of the specimens from routine clinical diagnosis could have led to over-estimation of genotyping failure rates. Also, our analyses did not involve paired plasma and DBS specimens and as such our comparisons could have been influenced by other demographic and clinical variations in individuals from whom the specimens were collected. Despite these shortcomings, the findings herein remain applicable to other laboratories in our setting and this study will set precedence for further research into strategies of improving genotyping rates. It is also worth noting that our study strength was the use of a large sample size of specimens analysed in our laboratory and the inclusion of both samples from research studies and non-research focused sources. To the best of our knowledge, this is the first study in our setting to determine the laboratory genotyping success rates and examine the correlates of genotyping failure of unpaired DBS and plasma specimens from multiple studies/sources.
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