The Effectiveness of HIV Drug Resistance Mutation Testing on Dried Blood Spot (DBS) Samples in Vietnam

Tram Hong Tran¹, Thien Huu Doan¹, Huong Thi Thu Phan², Xuyen Huu Doan^3, Thai Duy Nguyen^{1, *}

¹National Institute for Control of Vaccines and Biological, 1 Nghiem Xuan Yem Str., Dai Kim Ward, Hoàng Mai Dist., Hanoi, 100000

²Vietnam Administration of HIV/AIDS Control, Lane 8 Ton That Thuyet Str., My Dinh Ward, Nam Tu Liem Dist., Hanoi, 100000

³Vietnam University of Traditional Medicine, 2 Tran Phu Rd., Mo Lao Ward, Ha Dong Dist., Hanoi 100000

*Corresponding author: Nguyen Duy Thai, National Institute for Control of Vaccines and Biologicals, 1 Nghiem Xuan Yem Str., Dai Kim ward, Hoàng Mai Disst., Hanoi, Vietnam, 100000

Received: 17 September 2023; Accepted: 29 September 2023; Published: 06 October 2023

1. Introduction

Worldwide, dried blood spot (DBS) samples have been extensively studied for early diagnosis in children under 18 months suspected of HIV infection [8, 9], measuring HIV viral load  (9-19) and identifying drug-resistant HIV mutation genotypes [11, 20-45]. However, DBS contains both pre-viral DNA and cell RNA, which can lead to false positives, particularly in cases of plasma samples with very low viral loads or undetectable. Therefore, the World Health Organization (WHO) recommends cautious consideration when using DBS for viral load testing [11, 19]. In addition, the WHO has collaborated with reputable testing laboratories to develop, confirm, and standardize methods for detecting drug-resistant HIV mutations from DBS samples [15].

In Vietnam, the method of collecting samples on dried blood spot papers is widely applied in medical practice for diagnosing infectious diseases, metabolic disorders, hereditary conditions, and more in newborns and young children due to its advantages in terms of time, convenience in sampling, preservation, and sample transportation. It has also been used for early diagnosis of HIV infection in children under 18 months since 2009 [46], measuring HIV viral load from DBS samples since 2016 [47-50] and conducting genetic sequencing studies on DBS samples compared to plasma samples to identify drug-resistant HIV mutations since 2012 [51].

Testing for HIV-1 drug resistance mutations is an essential part of WHO's global drug resistance evaluation and prevention strategy [52, 53]. Plasma samples are always considered the gold standard for identifying the HIV drug resistance genotype. However, the use of plasma samples faces many challenges in terms of personnel and equipment needed to preserve the samples, especially in impoverished rural areas, remote and isolated regions, particularly in low- and middle-income countries. Therefore, dried blood spot (DBS) samples have been and are being used as a replacement for plasma samples in determining the HIV drug resistance genotype. The DBS sampling technique is simple, with a small sample volume (100ul/drop), can be stored at room temperature, has minimal cross-contamination, is easy to transport, requires no special laboratory handling, and can be used for routine clinical or surveillance purposes [54]. Mac dù có nhung ?u diem nhu vay nhung DBS van có mot so nh??c diem, truoc het là do nhay cua Despite these advantages, DBS has some limitations, primarily the sensitivity of the viral RNA amplification process is less than with plasma samples due to the smaller volume. Additionally, for patients with a low viral load, the genotype results from DBS samples may not accurately reflect the current drug resistance mutations, so it is recommended to use plasma samples for testing in these cases. The virus's RNA in DBS can degrade if the DBS gets moist or is stored at room temperature for an extended period (> 14 days), it should be kept in a deep freezer (-70°C) immediately after the sample dried and before testing with this case. Thus, our research aims to compile and evaluate the efficacy of various HIV drug resistance mutation testing products on DBS samples compared to traditional plasma samples. This would allow for the selection of appropriate testing kits for detecting HIV drug resistance mutations in DBS samples in mountainous areas, remote regions, borders, islands, and other areas in Vietnam where the conditions for using other plasma samples are not met.

2. Method

We conducted a search for open-access articles on databases including PubMed, Scopus, Web of Science, and Google Scholar from 2006 to the present using the keywords: Drug resistance mutation testing; HIV gene sequencing assays; HIV testing by dried blood spot (DBS); HIV testing on plasma samples. A total of 5,150 articles were found for these keywords. After filtering based on criteria that combined HIV drug resistance mutation testing on dried blood spot samples and plasma samples, we selected 56 relevant reports for analysis in this study. The protocol for this assessment follows the PRISMA guidelines.

Figure 1: Flow chart for determination of selected studies

3. Results

3.1. Evaluation of HIV drug resistance mutation testing products on DBS samples

Since 2004, following the recommendation of WHO, low and middle-income countries have optimized techniques and implemented dried blood spot (DBS) samples in HIV drug resistance mutation testing. By 2020, four main products were used worldwide (Table 2)  [10, 12-14, 20-25, 27-29, 35-39].

Table 1: Global studies on HIV gene testing techniques using dried blood spot (DBS)

The results in Table 1 indicate that there are four main products used globally for HIV drug resistance mutation testing: TruGene, ViroSeq™, ATCC kit, and In-house nested RT-PCR. These products are designed to sequence the Pol gene region, encoding the amino acid substitutions known to cause ARV drug resistance[7]. The products TruGene, ViroSeq™, and ATCC kit (Thermo Fisher) are commercial offerings, of which TruGene is no longer available in the market due to its outdated equipment. Given that countries with high HIV rates are predominantly poor, the cost of these products is a significant concern. Therefore, to align with economic conditions, the In-house nested RT-PCR technique has been researched and widely applied for gene sequencing to detect HIV drug resistance mutations [10, 12-14, 20-22, 25-28, 35-38]. Out of the 18 studies listed in the table, 15 utilized the In-house nested RT-PCR technique. In Vietnam, DBS samples are stored at room temperature for 2 to 4 weeks and then preserved at -20°C until testing. Treatment failure samples are selected for sequencing to detect HIV drug resistance using the In-house nested RT-PCR method provided by the French National Agency for Research on AIDS and Viral Hepatitis (ANRS) [10].

3.2 Effectiveness assessment of DBS samples compared to plasma samples

For DBS samples, in order to have a foundation for widespread and convenient deployment in the field, experts have conducted several tests to study the stability of DBS samples when stored at different temperatures. According to the WHO summary in 2020, 8 studies have been carried out (Table 3) [7].

Table 2: Stability of DBS samples at various temperatures

Research results from Table 2 indicate that the sample remains stable when stored at room temperature, or at 4°C for up to 2 weeks, or at -20°C for 2-3 years, or at -30°C and -70°C for extended periods. Based on these studies, the WHO has provided guidelines for transporting DBS samples [7]. In Vietnam, following the success of the research on DBS samples [47-49], the Ministry of Health issued Decision 1112/QD-BYT providing guidelines for conducting HIV load testing in monitoring and treating HIV/AIDS. Specifically, in Annex 3B, the procedure for sampling, storing, packing, and transporting DBS samples for HIV load testing was stipulated, similar to the WHO's procedure as below [55].

Figure 2: Diagram of transporting DBS samples according to WHO recommendations

Research indicates that DBS sampling is simple, manpower-efficient, labor-saving, and convenient for transportation. It can be transported at normal temperatures without a complicated cold chain like plasma sample storage. Moreover, it also doesn't break and cause cross-contamination. From a review of 18 studies worldwide on gene sequencing tests to detect HIV drug resistance mutations, eight studies did not compare mutation locations between DBS samples and plasma samples but were conducted directly on HIV-positive DBS samples [13, 21, 22, 27, 29, 36, 37, 39]. The remaining ten studies compared mutation locations between DBS samples and plasma samples, as shown in Table 4 [10, 12, 14, 20, 23-25, 28, 35, 36, 38].

Results in Table 3 indicate that, depending on the specific product and chemicals used, the sensitivity in detecting mutations varies at different HIV viral load thresholds. All studies reported results that for samples with a viral load > 1.000 copies/ml, the amplification success rate is higher than for those with a viral load < 1.000 copies/ml (>70%). Consequently, the WHO has recommended that for DBS samples, the minimum viral load threshold should be 1000 copies/ml (with the viral load value taken from measurements on plasma samples) for sequencing tests to detect HIV drug resistance mutations [7]. In Vietnam, the threshold for treatment failure in HIV is set at ≥ 1.000 copies/ml, as stipulated in professional guidelines and legal documents [55].

Table 3: Amplification success rate with HIV-positive DBS sample

Table 4: Comparison of gene sequencing between DBS samples and plasma samples

Table 4 shows that in 10 studies comparing the similarity in sequencing success rates between DBS samples and plasma samples, the results are equivalent, averaging about 97%. This provides a basis for underdeveloped countries to widely implement HIV molecular biological testing. In Vietnam, there are two studies comparing the genetic sequencing results between DBS and plasma. One by Monleau et al reported a 78% similarity in mutation positions [10]. Meanwhile, the study by Zhou et al showed a similarity rate of up to 98.9% in mutation positions [35].

4. Discussion

Results in Table 2 showed three utilized commercial products TruGene, ViroSeq™, and the ATCC kit (Thermo Fisher). The remaining 15 studies used the In-house nested RT-PCR technique. These studies were primarily conducted in poorer, underdeveloped countries such as Africa, Vietnam, Thailand, etc. With a high number of HIV-infected individuals and low economic status, especially in African regions, there is a scarcity of medical manpower and equipment. Thus, using commercial products to detect HIV drug resistance is costly and challenging. Hence, implementing drug resistance mutation testing using the In-house nested RT-PCR technique is more fitting for these underdeveloped countries' realities [10, 12-14, 23, 27, 35, 36, 39].

Plasma samples have always been considered the gold standard in HIV molecular biological tests. However, since 2009, the world has researched the use of dried blood spots (DBS) for early diagnostic testing of HIV in children under 18 months using PCR technology. WHO recommended its use in countries with high HIV prevalence such as Africa, Vietnam, Thailand, etc [8, 46]. Manufacturers like Abbott, Roche, and others have since developed commercial kits to expand its use for HIV viral load testing and resistance mutation detection in DBS samples. Yet, implementing DBS for viral load testing and seeking resistance mutations in HIV has faced challenges, especially for samples with low viral loads of <1000 copies/ml [35]. In the study by Zhou et al., there was a focus on optimizing genotypic testing using the low-cost In-house technique to monitor and track HIV drug resistance in underdeveloped countries with high HIV prevalence such as Africa, Vietnam, Thailand, etc [35]. Additionally, the research also explored the application of DBS samples in drug resistance testing as an alternative to plasma samples. The study of Zhou et al. had indicated [35].

The optimized In-house testing method demonstrated high sensitivity suitable for various HIV-1 subtypes such as M and CRF groups were carried out on both plasma and DBS samples collected from six countries with high HIV prevalence, namely Cameroon, Malawi, Nigeria, Zambia, Thailand, and Vietnam. This In-house testing, when compared with commercial kits like ViroSeq™ and Trugene, yielded equivalent results, with respective accuracies of 99.3%, 99.6%, and 99.1%. Moreover, the research team estimated that the reagent cost per test for the In-house method is 40 USD, compared to 213.20 USD for Trugene and 172.86 USD for ViroSeq™. Hence, both in terms of technical sensitivity and economy, the In-house nested PCR technique can be widely implemented for HIV drug resistance testing in underdeveloped countries for patients suspected of treatment failure with viral loads >1000 copies/ml.

On the other hand, the study successfully compared the results of genotypic sequencing to identify drug resistance mutations between DBS samples and plasma samples. The HIV genotypic sequencing results on 18 out of 26 DBS samples compared to their corresponding plasma samples collected from virologically failing patients in Nigeria and 69 out of 72 DBS samples compared to their corresponding plasma samples collected from patients suspected of treatment failure in Vietnam were identical in terms of drug resistance mutation points. Notably, the 18 DBS samples from Nigeria were stored at room temperature for an average of 85 days before being transferred to the laboratory. Additionally, two external quality control DBS PT samples, whether shipped frozen or at ambient temperature, resulted in successful amplification and genotypic sequencing, except for one sample with the lowest HIV load (<1.000 copies/ml) that was transported at ambient temperature. These results suggest that the optimized In-house nested PCR test has high sensitivity in determining the genotype for both plasma and DBS samples. Hence, the study indicates that DBS samples remain stable even when stored at room temperature for extended periods. However, they need to be meticulously packaged and stored in an air-conditioned room with low humidity (possibly with the aid of a dehumidifier) to ensure better sample quality. Proper packaging and storage of DBS are crucial factors to guarantee successful genotyping results. The research also suggests that for samples with low viral loads, the packaging and transportation must be even stricter to maintain sample quality. In cases where the DBS sample cannot be amplified, it is imperative to use plasma samples.

Another study by Zhang et al. indicated that DBS samples have advantages such as not requiring expensive equipment, easy for training, not necessitating cold chain storage, and not contaminating the surrounding environment, etc (39). The study also estimated that the cost of transporting DBS samples is about 250 USD depending on the country, while the estimated cost of using plasma samples is approximately 5,000 USD. Furthermore, the use of the ATCC kit (essentially a commercialized version of the In-house nested RT-PCR by Thermo Fisher) also reduces the cost of determining the HIV drug resistance genotype. Other HIV drug resistance genotyping products available in the market cost around 200 USD per test at the time of the study, while the cost of testing with the ATCC kit is about 60 USD per test. For 500 samples genotyped for HIV, just the cost of the drug resistance testing kit alone saves 50.000 USD. Based on these credible research results, WHO has recommended that developing countries like Vietnam use the In-house nested RT-PCR technique on DBS samples for HIV drug resistance mutation testing, as per the WHO 2020 guidelines [7].

5. Conclusion

The In-house test ensures efficiency and cost savings in identifying drug-resistant HIV genotypes compared to commercial kits. DBS samples are cost-effective, easy to train, and do not require cold storage like plasma samples. Also, according to WHO recommendations, dried DBS samples are safe and non-infectious, making them suitable for areas with limited medical resources. Thus, using the In-house technique on DBS samples is effective for HIV testing to mitigate the growth and transmission of drug-resistant HIV in the community. However, for samples with a viral load of less than 1,000 copies/ml, plasma samples are the best choice.

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