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Towards virological monitoring of HIV-1 drug resistance in resource-limited
settings
Aitken, S.C.
Publication date
2014
Link to publication
Citation for published version (APA):
Aitken, S. C. (2014). Towards virological monitoring of HIV-1 drug resistance in
resource-limited settings.
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General discussion
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fully active antiretroviral (ARV) regimen can quickly and efficiently suppress virus replication, allowing the immune system to reconstitute, preventing disease progression and AIDS-related death. While improving access to treatment is key to relieving the global HIV-1 burden, decrease virus transmission(1) and increase life expectancy, good quality supportive careis necessary for sustainability of HIV treatment success. Such appropriate quality of care should ideally include virological monitoring of patients, for example, regular monitoring of viral suppression and eventually, in certain cases, HIV drug resistance (HIVDR) genotyping.
Simple and Affordable Monitoring
Scale-up initiatives have improved access to ART for millions of HIV-1 infected individuals in resource-limited setting (RLS), primarily due to major cost reductions of ART resulting from relentless lobbying by individuals and activists in NGO’s, governments, UN agencies and the private sector. However, improvement of immunological and virological monitoring has not followed suite. In 2008, a consortium of public and private members from The Netherlands, Belgium, Luxembourg, and South Africa joined forces in the Affordable Resistance Test for Africa (ARTA) project with the aim to develop a comprehensive set of affordable tools to enable HIV-1 monitoring in Africa. Components of this initiative included a virological failure assay (VFA) for virological monitoring, and subtype-independent confirmatory HIVDR genotyping assays, all of which are suitable for use with dried blood spot (DBS) samples.
The Need for Virological Monitoring
The use of immunological and clinical monitoring has previously been shown to be insufficient for HIV-1 treatment management (2-6), resulting in unnecessary
therapy switching or extended exposure to a failing regimen. Virological failure (VF) generally precedes immunological failure(7), and early detection
of VF through regular VL monitoring allows for timely intervention and could prevent accumulation of drug resistance mutations (DRMs) resulting from
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prolonged viral replication in the presence of a failing treatment regimen(5, 8). Previous studies have shown a correlation between lack of virological
suppression and emergence of HIVDR(9-16), making regular VL determination
the tool of choice for preventing accumulation of HIVDR mutations. Recently, the WHO have updated their guidelines on monitoring the response to cART, recommending VL testing (at 6 months after ART initiation and every 12 months thereafter) as the preferred monitoring approach to diagnose and confirm ARV treatment failure(17).
There are currently numerous commercial assays available for VL monitoring using an individual’s plasma, which are being performed at regional and reference laboratories. Such laboratories often receive vast numbers of samples for testing, and turn-around time for reporting results can often be lengthy, mainly due to long transport times or distances and complicated sample logistics.
Point-of-care (POC) assays for VL may be a good way to provide rapid results when and where they are needed, subsequently improving retention into care. There are currently no commercially available POC VL tests in routine use, although several platforms are in development and may be launched shortly(18). Most POC VL tests use all-in-one cartridges to which
a single sample or a limited number of samples are added and processed. Most systems are designed for use with whole blood obtained from finger- or heel-prick, but some are also compatible with plasma and serum. In general, results are available after 30 minutes up to two hours, which may still not be optimal for busy clinics. The major drawback of these assays may be the limited throughput of the POC systems, given the high burden of infected individuals in areas where such tests are needed the most. Furthermore, the proposed costs of POC tests are comparable to current commercial VL tests. Drawbacks to POC testing include service and maintenance of the machines at remote locations, reagent stock-outs, availability in clinics, and the need for training on use to high numbers of laboratory technicians or health care
workers.
Another approach to improving access to VL testing is large central facilities geared up for super-high-throughput processing. Such an approach will have similar drawbacks to POC testing, but to a lesser degree, as there will be fewer centres that need training and support. These centres would most likely be located in urban settings where access to technical support and regular reagent shipments are available. However, in many cases, the cold-chain dependent transport of samples to testing facilities still remains a problem, especially if samples for testing are plasma. The use of DBS sampling may improve this, and their use with commercial assays has been demonstrated(19, 20), however validated commercial assays for DBS VL testing
are still not available.
Commercial VL assays provide precise measurement of viral RNA copies present in a sample, but are generally costly and technically complex. In many cases exact VL quantitation is not required to determine treatment failure. Therefore, an assay that classifies a sample as either above or below a treatment success threshold could have wide application. Chapter 2 describes
a virological failure screening assay (VFA) based on an open-platform real-time PCR, that was designed and piloted by the ART-A consortium. The evaluation of the VFA for plasma and DBS samples determined the lower limit of detection to be 1.00E+03 and 5.00E+03 copies/ml, respectively, which is in concordance with the current WHO guidelines(17). A comparison
of results generated using the VFA showed good correlation with VL results previously determined using commercial VL assays. The unique aspect of the described assay, in comparison to previously described laboratory developed virological failure assays, is its multiplex design enabling detection of an internal control in each sample, ensuring accurate and reliable results from isolation to amplification. The assay can be performed on a compact, low-cost real-time system for use in regional laboratories, as well as on larger, high-throughput systems at reference laboratories. We demonstrated its
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versatile use by application in RRS as part of the development, followed by successful application in RLS settings in regional and reference laboratories in Uganda (Chapter 2) and South Africa (Chapter 4).
Our approach of a virological screening assay (Chapter 2) falls between POC
and centralised facility testing. It is designed for use in regional laboratories servicing a number of nearby clinics, preventing long distance transport of samples. The VFA is capable of parallel processing more samples than POC VL tests, but requires more training and the availability of laboratory facilities suitable for performing molecular techniques. The open-platform design of the assay makes it adaptable to already equipped laboratories. Reagent costs for the assay are country dependent, with a per sample cost, based on a run of ten samples including controls, of 22.00 USD calculated for UMCU, The Netherlands, and 27.00 USD for JCRC, Uganda. Like POC and commercial VL assays, the VFA is still subject to reagent stock-outs, servicing, and need for training. Unfortunately, the cost is not sufficiently low to warrant its use in regular patient monitoring, but could still be used in DBS based populations studies, which is currently under evaluation in Uganda(21). Highly significant
cost reductions should be achievable with increased application of the assay by laboratories.
Surveillance and Monitoring for HIVDR
One of the key statements made in Getting To Zero is “know your epidemic, know your response”, which, according to the aims of the WHO, refers to knowing which population groups are most at risk(22). However, in addition
to this, an important aspect of the HIV epidemic that is key to sustainable treatment using first-line therapy is knowing the types and prevalence of HIVDR within the population. This can be achieved through surveillance and monitoring of HIVDR by genotyping. Knowing the prevalence and types of HIV DRMs in the infected population of a country can help health policy makers to make informed decisions about choices of treatment regimens and sequencing of first- and second-line drugs. In RLS, HIVDR genotyping
for individual patient monitoring is not yet possible due to costs and technical complexity, though may be of increasing importance with the improvement of treatment coverage and duration in these countries. In a prospective study of individual patient management for HIVDR in individuals accessing failing ARV therapy (Chapter 7), it is demonstrated that two thirds of patients
receiving a failing ARV regimen, identified using virological monitoring, indeed harboured resistance mutations to a first-line regimen. This is comparable to the findings of the WHO surveillance activities for acquired HIVDR(23).
The development of laboratory developed, so called “in-house”, HIVDR genotyping assays has become popular over the years, initially due to suboptimal sensitivity of commercial assays for non-B subtypes(24, 25), but
more recently to overcome high costs of currently available commercial assays. Both commercial and laboratory developed HIVDR genotyping assays generally target the protease (PR) and reverse transcriptase (RT) regions of HIV either in separate nested RT-PCR reactions for each region(26-28), or alternatively amplifying combined PR-RT regions either in a
two-part RT-PCR(25) or together in a nested RT-PCR(29). Targeting both PR
and RT in a single fragment reduces the number of amplification reactions required, in turn reducing the costs, and risk of contamination or samples mix-up, however can also negatively affect the sensitivity of the assay. Typical laboratory developed assays have genotyping success rates for PR and RT ranging from 88-97%, with assay sensitivity having levels of detection (LOD) between 5.00E+02-1.00E+03 RNA copies/ml. Whilst some assays are still optimised for HIV-1 subtype-B samples(27, 29), assay compatibility with non-B
subtypes is now greatly improving(25, 26, 28).
In chapter 4 we describe the developed and evaluation an assay for
assessing PR and RT drug resistance using a single fragment nested RT-PCR approach. The assay displays sensitivity and specificity for the major HIV-1 group-M subtypes and CRFs at the highest success range compared to any other published alternative assays (~98%).The assay is universally
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applicable with equal specificity for group M HIV-1 subtypes with a sensitivity of 1.00E+03 copies/ml for plasma and DBS samples. This assay has met the acceptance criteria of the Virology Quality Assessment (VQA)/WHO genotypic HIVDR 2013 DBS proficiency panel (GEN002BS; VQA Program, Rush Medical Centre, Chicago, USA). Further testing is currently underway to meet the official WHO validation criteria. Application of the assay in RLS based studies has been successful, both for plasma (Chapter 6) and DBS
samples (Chapter 7).
Whilst the PR-RT HIVDR genotyping assay performs excellently, genotyping the PR region for the most part is excessive, in particular when cART is solely based on a combination of RT inhibitors (RTIs). Current ART regimens in RLS(17) are still primarily based on first-line, RTI based, therapy, and as
such an HIVDR genotyping assay that analyses the RT region only would be effective for determining the presence of resistance conferring mutations . All major mutations affecting efficacy of reverse transcriptase inhibitors, as defined by the IAS drug resistance mutation list, are located between RT amino acids (aa) 41 and 238 (30). An in silico analysis of HIVDR genotyping
profiles of this specific RT region demonstrated that the predicted drug susceptibilities were equally informative compared to sequences that more broadly cover the RT gene (aa1-400) (31). With this in mind, we developed
and evaluated an RT-only HIVDR genotyping assay (Chapter 3).
Unlike currently available HIVDR genotyping assays, the RT-only assay only amplifies the region of RT encompassing all HIV-1 DRMs relevant to current first-line ART in a single RT-PCR using primers carefully designed and selected to cover all major HIV-1 group-M subtypes and circulating recombinant forms (CRFs). Subsequent sequencing is performed using only a single forward and a single reverse primer, compared to four to six primers needed for commercial and in-house assays (25, 26, 28, 32). Decreasing the number
of reactions required for amplification and sequencing in turn decreases the overall cost of the assay as well as minimizes assay complexity, hands-on
time, contamination risk, and turnaround time. The assay has demonstrated a genotyping success rate of 95% and 84% with a VL of ≥5.00E+03 and ≥1.00E+03 RNA copies/ml, respectively.
The single RT-PCR design of the assay makes it ideal for application in RLS where there is often only basic laboratory infrastructure, making it difficult to prevent contamination often encountered with nested PCR assays. The successful use of the assay has been demonstrated in reference laboratories in Uganda (Chapter 3) and South Africa (Chapter 4). The assay is also suited
for use with DBS as the short fragment size ensures reliable amplification even for samples that have been exposed to ambient temperatures for up to two weeks during transport and storage, (Chapter 8). Application of the
assay on the 2013 VQA panel was very successful, with comparable results to the PR-RT assay, and is also being validated to meet the WHO criteria. However, it is not easy to compare to the results to other assays as this RT-only approach is unique to the assay described in Chapter 3.
Toward virological monitoring in RLS: The ARTA Algorithm
The components of the ARTA project described in this thesis fits together in a algorithm that may be applicable for HIV virological monitoring of HIV infected individuals in RLS (Figure 1). Improving virological monitoring in RLS starts at the level of sample collection. By using DBS sampling, virological monitoring can be made accessible in remote clinic settings, using finger/ heel-prick collection, and intermediate clinics using venipuncture or finger/ heel-prick. Collected DBS can be sent at ambient temperature (Chapter 8) to
a regional laboratory with basic molecular technique capabilities or reference laboratories with more extensive molecular technique capabilities, where virological failure screening can be performed with the VFA (Chapter 2).
Results are sent back to the collection site, as either suppression or failure. In the event of VF, adherence counseling should be intensified to control for sub-optimal adherence, and another VFA screen should be performed 6-8 weeks later. Should the second result indicate continued virological failure,
9
the remaining DBS sample can be processed by a reference laboratory for HIVDR genotyping (Chapter 3 and 5). HIVDR genotyping results can be
analyzed and using the ARTA-developed sequence analyses software(33) and
interpreted for optimized treatment prior to being reported to the clinic for further interpretation. If the HIVDR genotyping result indicates the presence of HIVDR, a clinician can make an informed decision on the most effective course of treatment to ensure viral suppression. Should the result indicate no HIVDR, poor adherence should be addressed with the individual, which may avoid an unnecessary therapy modification.
Figure 1. The ARTA algorithm for HIV virological monitoring in RLS.
Money, money, money
General application of VL monitoring in RLS is restricted by cost and technical requirements. For example, scaling up of facilities for VL testing requires large initial funding in order to buy equipment and reagents, and employ and train appropriate technicians. However, a recent study by Hamers et al. on the cost-effectiveness of laboratory monitoring using VL and CD4 counts in RLS has shown that the costs associated with such tests are balanced out by preventing unnecessary switching from first-line therapy to more costly second-line therapy(34). The study has shown using Markov
modelling that performing VL or CD4 counts at 6-month intervals would incur similar costs, whilst when performed every 12 months CD4 counts were more cost-effective, though both methods yield cost savings for long-term ART management compared to a symptom-based diagnostic approach(34).
Whilst CD4 monitoring my be slightly less expensive, regular VL testing to determine therapy failure is the test of choice as it is more informative than CD4 counts(35-39).
One approach of increasing the affordability and scalability of molecular diagnostics in Africa is using in-house assays that are “open platform”: all primer sequences and protocols are openly accessible and the assay can use various equipment and reagents that can be ordered from multiple manufacturers. The Southern African Treatment Resistance Network (SATuRN) and ARTA are key supporters the use of open platform tests. SATuRN has negotiated discounted reagents and technical support with reagent providers in order to decrease the cost and increase access of HIVDR genotyping in Africa (http://www.bioafrica.net/saturn/). The described VF screening assay (Chapter 2) and HIVDR genotyping assays (Chapter 3) have the potential to use the same approach. In addition, SATuRN and
ARTA have been providing extensive training on the usage of molecular diagnostics for treatment monitoring with over 1,500 physicians, nurses and health care workers trained in Africa. These organization training platforms can be used to expand and support the usage of the VF screening (Chapter 2) and HIVDR genotyping assays (Chapter 3) in Africa.
Compared to VL testing, HIVDR genotyping requires more sophisticated equipment and technical expertise, and is restricted to reference laboratories. With the use of laboratory developed assays such as the described RT-only HIVDR genotyping assay, pragmatic HIVDR testing could be performed for HIV-infected individuals identified with VF using VL monitoring. From a reagent perspective, using the described assay would result in a >75% saving compared to using a commercial assay such as ViroseqTM Genotyping System
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2.0 (Celera Diagnostics, USA), and approximately a 40% saving compared to using our in-house PR-RT HIVDR genotyping assay(32). Furthermore,
the shorter laboratory protocol and sequence to be analysed, results in a lowering of labour compared to currently available methods (25, 28, 32). Like VL
monitoring, strategic use of HIVDR genotyping could also potentially be cost neutral by preventing unnecessary switching to more expensive second-line regimens, as was demonstrated by Rosen et al. using Markov modelling(40).
A newer trend is using next generation sequencing (NGS) to process more samples per run and at a lower cost(41, 42). Although NGS enables bulk
sequencing, the data that is generated is often extensive and complex. Recent developments in software have been made at the South African National Bioinformatics Institute (SANBI) that allow for efficient and sensitive analysis of HIVDR NGS data(43), which could greatly improve HIVDR surveillance by
allowing more samples to be processed and at a lower cost. The two current leading NGS technologies for virus sequencing applications include 454 by Roche which generates reads of approximately 500 bases, and the MiSeq by Illumina which has read lengths of approximately 250 bases. The described RT-only assay could easily be adapted for use in NGS, especially due to the short target fragment.
In addition to laboratory-based techniques, computational models for predicting virological response to ART for patients could serve as an alternative. Such a model has been developed by the HIV Resistance Response Database Initiative (RDI) group with initial results for application in RRS showing comparative outcomes to using HIVDR genotyping(44-46).
The model is currently being adapted for application in RLS, with initial data showing good application(47). Whilst this could avert the need to individual
patient monitoring using HIVDR genotyping, VL monitoring is vital for each patient, and surveillance of HIVDR is still necessary to adapt the model to region use(48).
Final Summary
By the end of 2011 more than 6.6 million infected individuals in RLS had access to ART (49). With the new criteria for ART initiation added to the 2013
WHO guidelines on HIV treatment an additional 9.2 million people qualify for ART compared to the 2010 guidelines(50), bringing the estimated number of
people eligible for ART to 25.9 million in 2013(50). In addition to improving ART
staring criteria, the quality of patient monitoring has also been addressed by including VL monitoring as a strong recommendation for patients receiving ART(17). In addition to currently available commercial VL monitoring tools,
there are several new assays in the pipeline, including POC VL testing and VF screening assays as has been described in this thesis. VL monitoring assays that are compatible with DBS sampling will be the most effective way to provide access to VL monitoring in RLS. In addition to VL monitoring, surveillance and monitoring of HIVDR is playing an important role in ensuring long term use of first-line ARVs. Current population based HIVDR monitoring is providing valuable data to further understand the development of DRMs, which can be subsequently used to guide choices for ARV use in RLS, as well as for resistance modelling studies. Although individual patient HIVDR genotyping is not yet available, using DBS sampling in conjunction with pragmatic HIVDR genotyping may promote feasibility in the future.
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