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Towards virological monitoring of HIV-1 drug resistance in resource-limited
settings
Aitken, S.C.
Publication date
2014
Document Version
Final published version
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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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-1 drug resistance in resource-limited settings
Susan C.
Aitken
Towards virological monitoring
of HIV-1 drug resistance
in resource-limited settings
Susan C. Aitken
HIV-1 drug resistance
in resource-limited settings
Copyright © 2013, Susan C. Aitken, The Netherlands. No part of this thesis may be reproduced, stored or transmitted in any form or by any means, without prior permis-sion of the author.
ISBN: 978-94-6108-578-8
Layout and cover design: Susan Aitken and Janna McCall-Peat Printed by: Gildeprint Drukkerijen
The studies included in this thesis were part of the Affordable Resistance Test for Africa (ART-A) program and were supported by a grant from the Netherlands Or-ganization for Scientific Research for Global Development (NWO/WOTRO), under the Netherlands African Partnership for Capacity Development and Clinical Inter-ventions against Poverty-related Diseases (NACCAP; grant W.07.05.204.00), The Hague, The Netherlands.
HIV-1 drug resistance
in resource-limited settings
ACADEMISCH PROEFSCHRIFT
ter verkrijging van de graad van doctor aan de Universiteit van Amsterdam op gezag van de Rector Magnificus
prof. dr. D.C. van den Boom
ten overstaan van een door het college voor promoties ingestelde commissie, in het openbaar te verdedigen in de Agnietenkapel
op woensdag 22 januari 2014, te 10:00 uur
door
Susan Claire Aitken
Promotores: Prof. dr. T.F. Rinke de Wit Prof. dr. W. Stevens
Co-promotores: Dr. R. Schuurman Dr. C.L. Wallis
Overige leden: Prof. dr. M.D. de Jong Prof. dr. P.R. Klatser Prof. dr. J.M.A. Lange Dr. M. Nijhuis
Prof. dr. P. Reiss Prof. dr. E.J. Wiertz
Abbreviations General Introduction
Development and evaluation of an affordable real-time qualitative assay for determining HIV-1 virological failure in plasma and dried blood spots. Journal of Clinical Microbiology (2013) 51(6):
1899-905
A pragmatic approach to HIV-1 drug resistance determination in resource-limited settings using a novel RT-only genotyping assay.
Journal of Clinical Microbiology (2013) 51(6): 1757-61
Evaluation of an affordable HIV-1 virological failure assay and an-tiretroviral drug resistance genotyping protocol. Journal of
Virolog-ical Methods (2013) 194(1-2): 300-7
Development and evaluation of an assay for HIV-1 protease and reverse transcriptase drug resistance genotyping of all major group-M subtypes. Journal of Clinical Virology (2012) 54: 21-25 Accumulation of drug resistance and loss of therapeutic options precede commonly used criteria for treatment failure in HIV-1 sub-type C infected patients. Antiviral Therapy (2012) 17(2):377-86 HIV-1 resistance testing on dried blood spots enables individual patient management in rural South African Setting. Unpublished Stability of 1 nucleic acids in dried blood spot samples for HIV-1 drug resistance genotyping. Unpublished
General discussion Summary
Nederlandse samenvatting Authors and affiliations Acknowledgements List of publications About the author
Chapter 1 Chapter 2 Chapter 3 Chapter 4 Chapter 5 Chapter 6 Chapter 7 Chapter 8 Chapter 9 Appendix 6 11 36 61 81 107 125 151 163 183 205 211 217 221 227 233
aa amino acid
AAC accelerated access initiate ABC abacavir
AIDS acquired immunodeficiency syndrome
AIGHD Amsterdam institute for global health and development AMC academic medical centre (Amsterdam)
(c)ART (combination) antiretroviral therapy ARTA affordable resistance test for Africa ARV antiretroviral (drug)
ATV atazanavir AZT zidovudine
cDNA complementary DNA
CCR5 chemokine (C-C motif) receptor 5 CBV combivir
CI confidence interval
CRF circulating recombinant form Ct cycles to threshold
CXCR4 chemokine (C-X-C motif) receptor 4 CV (%) coefficent of variance
d4T stavudine DBS dried blood spot
ddI didanosine dideoxyinosine DLV delavirdine
DNA deoxyribonucleic acid DRM drug resistance mutation DRV darunavir
EDTA ethylenediaminetetraacetic acid EFV efavirenz
EMC encephalomyocarditis virus ENF enfuvirtide
ETR etravarine
EWI early warning indicator
FDA United States food and drug administration FPV fosamprenavir calcium
FTC emtrictabine GP glycoprotein
GRADE (HIV-1) Genotypic Resistance-Algorithm Deutschland GSS genotyping sensitivity score
HAART highly active antiretroviral therapy HIV human immunodeficiency virus HIVDB HIV database (Stanford DB) HIVDR HIV drug resistance
HIVResNet global HIV drug resistance network IAS international AIDS society
IC internal control IDV indinavir
IQR inter-quartile range
JCRC joint clinical research centre LED light-emitting diode
LIC low income country LOD level of detection LPV lopinavir
LTR long terminal repeat
MEGA molecular evolutionary genetics analysis MVC maraviroc
mRNA messenger RNA n number (of samples)
NACCAP Netherlands African partnership for capacity development and clinical interventions against poverty-related diseases
NCBI national centre for biotechnology information NA nucleic acid
NFV nelfinavir
NGO non-governmental organization NGS next generation sequencing NMC Ndlovu medical centre
NNRTI non-nucleoside reverse transcriptase inhibitor NPV negative predictive value
NRTI nucleoside reverse transcriptase inhibitor NVP nevirapine
NWO/WOTRO Netherlands organisation for scientifid reaseach for global development
PASER PharmAccess African studies to evaluate resistance PBMC peripheral blood mononuclear cells
PCR polymerase chain reaction (b)PI (boosted) protease inhibitor POC point-of-care
PMTCT prevention of mother-to-child transmission of HIV PPV positive predictive value
PR protease RAL raltegravir
RDI HIV resistance response database initiative RPV rilpivirine
RLS resource-limited settings RNA ribonucleic acid
RRS resource-rich settings
RT(I) reverse transcriptase (inhibitor)
RT-PCR reverse transcriptiase polymerase chain reaction RTV ritonavir
SANBI South African national bioinformatics institute SATuRN Southern African treatent resistance network SD standard deviation
SQV saquinavir mesylate
TAM thymidine analogue mutation TDF tenofovir
TDR transmitted drug resistance TPV tipranavir
UMCU university medical centre Utrecht UNICEF United Nations children’s fund
UNAIDS joint United Nations programme on HIV/AIDS USD United States dollar
VF(A) virological failure (assay) VL (HIV) viral load
WB whole blood
C
A
H
P
T
E
R
1
S
ince the first recorded cases in 1981, human immunodeficiency virus (HIV) has spread globally, resulting in the death of over 35 million people(1). The global HIV-1 epidemic has been estimated to include 34.0million (range 31.4-35.9 million) infected adults and children by the end of 2011. An alarming 69% (n= 23.5 million) of the total global infections are people residing in Sub-Saharan Africa. Prevalence is highest in adults aged 15-49, affecting 0.8% of the global adult population, and as high as 4.9% of adults residing in sub-Saharan Africa(2). People at greatest risk of
being infected with HIV are homosexual and heterosexual people having unprotected sex, sex workers, people who inject drugs, and children born to HIV-infected mothers(2).
HIV primarily infects CD4+ T-lymphocytes and macrophages, entering the body
via mucous membranes or through blood-blood contact. After transmission, an acute infection ensues, characterised by rapid virus replication and immune activation(3-5). The infection then stabilises to a chronic condition,
characterised by stable, continuous viral replication and a gradual decline in CD4+ T-lymphocytes(6). If not treated, the decline in CD4+ T-lymphocytes will
continue until the immune system of the infected individual is compromised, thereby making them vulnerable to opportunistic infections leading to acquired immunodeficiency syndrome (AIDS) and eventually death.
The number of people dying from AIDS-related illnesses was estimated to be 1.7 million (range 1.5-1.9 million) worldwide in 2011. Compared to 2005, this is a 24% reduction. The primary reason for this reduction is the scale-up and success of antiretroviral treatment (ART) programs, which have resulted in a decrease of AIDs related deaths and a decline in the incidence of new HIV infections. Antiretroviral (ARV) access in resource-limited settings (RLS) has also improved the health of people infected with HIV, enabling them to lead relatively normal lives, including being able to work and providing an income for themselves and their families(2).
1
Human immunodeficiency virus, HIV
HIV is an enveloped RNA virus belonging to the Family of the Retroviridae, genus lentiviridae. HIV-1 is the predominant global HIV species, with the less pathogenic HIV-2 found mostly in West Africa. HIV-1 is further divided into four groups, namely major (M), non-M/non-O (N), outlier (O) and pending the identification of further human cases (P), of which M is the primary contributor to the global HIV pandemic. Group M HIV-1 comprises of subtypes A- D, F-H, J and K, and numerous circulating recombinant forms (CRFs; currently n= 58), including common CRFs 01_AE and 02_AG(7, 8) (Figure 1). HIV-1
subtype C is responsible for the majority of global HIV-1 infections (48%), followed by subtypes A (12%) and B (11%). Evidence suggests that Central Africa is where the HIV epidemic originated and as such has the largest diversity of subtypes (Figure 1) (9-11). In sub-Saharan Africa infections are
primarily caused by HIV-1 subtype C, whilst in Europe and North America HIV-1 subtype B predominates, although subtype diversity has increased over the last decade, particularly in Europe(7).
Overview of the replicative cycle of HIV
During infection, HIV first binds to the surface of target cells by interaction of the viral surface glycoprotein 120 (gp120) with the CD4 receptor on the cell surface(12, 13). This binding causes a conformational change in gp120(14, 15), allowing for an additional interaction of gp120 with either CC chemokine
receptor 5 (CCR5)(16) or CxC chemokine receptor 4 (CXCR4)(17). This
interaction triggers the viral transmembrane subunit gp41 to interact with the target cell, eventually leading to fusion of the virus envelope with the target cell(18) and internalization of the virus particle. The virus uncoats, releasing
the genomic RNA into the cytoplasm of the cell. Once in the cytoplasm the genomic viral RNA is converted into double-stranded cDNA by the viral reverse transcriptase (RT), which is then complexed with integrase and other viral components to form the pre-integration complex(19) enabling entry
into the cells’ nucleus. In the nucleus, the integrase enzyme mitigates the integration of the double-stranded viral DNA into the host cell genome. Once integration has occurred proviral DNA will be copied into each daughter cell with every division cycle of an infected cell, thereby achieving long-term presence in the host genome. Furthermore, the proviral DNA serves as a template for transcription to produce viral mRNA and genomic HIV-1 RNA molecules. In the cytoplasm, viral mRNA is translated into viral precursor polyproteins, which are cleaved by protease (PR), a process necessary to produce infectious viral progeny(20). Cleaved viral proteins are then assembled
with RNA genomes into new virions on the cellular membranes(21), eventually
being released as virus particles (Figure 2).
Cells that have productive HIV replication generally have a short life span, either being destroyed as a result of virus replication or by the host immune response. HIV infection is not always productive, which is the case when infection stops at the point of integration and the cell enters a resting state, referred to as latently infected resting CD4+ T cells(23). Integrated proviral
DNA found in latently infected cells is referred to as archived virus, which can represent a viral population different to the currently circulating population.
1
Latently infected, resting CD4+ T cells are the largest barrier to curing HIV as they are a source for reactivation of viral replication(24-26).
Figure 2. Replication cycle of HIV (adapted from Engelman et al. 2012(22))
HIV Therapy
There are currently six classes of FDA-approved ARV drugs that target different steps of the HIV replication cycle: Nucleoside reverse transcriptase inhibitors (NRTI), non-nucleoside reverse transcriptase inhibitors (NNRTI), protease inhibitors (PI), fusion inhibitors, integrase inhibitors, and entry inhibitors (CCR5)(27). All currently available drugs for the treatment of HIV are
summarised with their introduction over time in Figure 3.
Initial treatment for HIV infection was based on the NRTI zidovudine (AZT) monotherapy. Although clinical benefit was demonstrated upon AZT treatment, the effect was rather temporal and lasted for only weeks to months(28, 29) due
Figure 3. Currently available FDA-approved antiretroviral drugs for HIV treatment,
with their introduction over time.
Nucleoside reverse transcriptase inhibitors (NRTI; Black): Zidovudine azidothymidine (AZT,
ZDV), didanosine dideoxyinosine (ddI), stavudine (d4T), lamivudine (3TC), abacavir sulphate
(ABC), enteric coated didanosine (ddI EC), tenofovir disoproxil fumerate (TDF), emtricitabine
(FTC). Non-nucleoside reverse transcriptase inhibitors (NNRTI; Blue): Nevirapine (NVP),
delavirdine (DLV), efavirenz (EFV), etravarine (ETR), rilpivirine (RPV), nevirapine extended
release (NVP-XR). Protease inhibitor (PI; Red): Saquinavir mesylate (SQV), ritonavir
(RTV), indinavir (IDV), nelfinavir mesylate (NFV), lopinavir and ritonavir (LPV/r), atazanavir
sulphate (ATV), fosamprenavir calcium (FPV), tipranavir (TPV), darunavir (DRV). Fusion
inhibitors (Green): Enfuvirtide (T-20, ENF). Entry Inhibitor (CCR5; Purple): Maraviroc (MVC).
Integrase inhibitor (Orange): Raltegravir (RAL), Elvitegravir (EVG). Cobicistat (COBI; pink)
is a cytochrome P450 3A enzyme inhibitor. Combination regimens are underlined. Generic drugs are shown in italics.
1981 1987 1991 1994 1995 1996 1997 1998 2000 2001 2003 2004 2005 2006 2007 2008 201
1
1986
First reported AIDS case First virus isolated
First FDA-approved blood test
AZT ddI d4T 3TC, SQV RTV, IDV, NVP NFV, DLV, AZT/3TC EFV, ABC LPV/r, ddI-EC, AZT/3TC/ABC TDF ENF, ATV, FTC, FPV MVC, RAL 2012
ABC/3TC, TDF/FTC, ddI generic
TPV, AZT generic DRV, TDF/FTC/EFV ETR RPV, TDF/FTC/RPV, NVP-XR EVG/FTC/TDF/COBI 1985 HIV named
First FDA-approved antiretroviral
1
to the emergence of viruses harbouring reduced susceptibility to the drug(30, 31). New drugs were developed, and in the early 1990s combination therapy
was shown to have a greater success for long term HIV treatment(32, 33). The
use of combination antiretroviral therapy (cART) was introduced in resource-rich settings (RRS) in the mid-1990s. With the introduction of cART, the number of people dying from AIDS-related illnesses substantially decreased
(34-36).
Antiretroviral Therapy Roll-out
When introduced, the cost of cART was on average USD 10 000 to 15 000 per person per year, restricting access to RRS. International lobby by HIV/ AIDS activists put pressure on governments and branded pharmaceutical companies to reduce the cost. In 2000 the International Aids Society (IAS) conference was held for the first time on African soil in Durban, South Africa. The conference highlighted the need for improved HIV treatment in RLS, and subsequently the Accelerated Access Initiate (AAI) was undertaken by the WHO, lowering the costs of cART considerably. In 2002 a total of only 300 000 infected individuals in low- and middle-income countries were receiving the necessary ART(1). The WHO “3 by 5” initiative(37) was responsible for
increasing access to cART to more than 6.6 million infected individuals in 2011(1). In 2011, a political declaration was made, the United Nations Political
Declaration on HIV and AIDS, where countries pledged to intensify efforts to eliminate HIV and AIDS(38). One of the goals of this pledge is to provide
antiretroviral therapy to 15 million people by 2015, and as a result just over 9.7 million infected individuals are currently receiving cART in low- and middle-income countries(39) (Figure 4 and 5).
The 2013 WHO guidelines on HIV treatment have greatly increased the number of people qualifying for treatment. People eligible for ART include all HIV infected adults and children older than five years of age presenting with stage 3 or 4 AIDS defining illnesses and/or with a CD4 count ≤500 (previously ≤350(41)) cells/mm3; with active TB infection; with HBV co-infection with
evidence of severe chronic liver disease; partners with HIV in serodiscordant couples to reduce HIV transmission (new); and pregnant and breastfeeding (new) women with HIV(40). For children younger than five years of age
infected with HIV, ART should be given regardless of WHO clinical stage or CD4 count. With the introduction of the new staring criteria, an additional 9.2 million people qualify for ART compared to the 2010 guidelines(41).
Figure 4. Actual and projected number of people receiving antiretroviral therapy in
low- and middle-income countries, by region, 2003-2015(39) (Source: 2013 Global
AIDS Response Progress Reporting (WHO/UNICEF/UNAIDS)).
When first-line ARV treatment is no longer effective in suppressing viral replication, in most cases due to emerging drug resistance, switching to a second-line regimen is warranted. The WHO suggested second-line regimen consists of a boosted PI, preferably atazanavir/ritonavir (ATV/r) or lopinavir/ ritonavir (LPV/r), and two NRTIs, preferably AZT and 3TC, the choice of which is dependant on an infected individuals previous first-line regimen(40).
In addition to use as a PI in second-line therapy, LPV/r and two NRTIs are also recommended in first-line treatment for HIV infected children younger
1
than three years of age(40). In general, the availability of PI-based regimens
varies largely between countries, mainly due to its much higher cost.
0 10 20 30 40 50 60 70 80 90 100 Angol a Botsw ana Burun di Cam eroon Chad Côte d 'Ivoir e Dem ocrat ic Repu blic of Cong o Ethiopi a Ghan a Keny a Lesotho Ma lawi Mozam bique Namib ia Nigeria Sout h Afric a Swa zilan d Ugand a* United Republ ic of Tanz ania Zam bia Zimbabw e
Figure 5. Antiretroviral therapy (ART) coverage among adults and children eligible for
ART in 21 African countries with a high burden of HIV infection, 2012. Blue: Southern Africa; Green: East and Central Africa; Yellow: West Africa. (Adapted from: 2013 Global AIDS Response Progress Reporting (WHO/UNICEF/UNAIDS) and 2013 UNAIDS/WHO estimates)
Current cART regimens for the treatment of HIV-1 in adults and adolescents in RLS, as recommended by WHO guidelines, are a combination of two NRTIs and one NNRTI. As this is the starting therapy, it is commonly known as first-line treatment regimen. The preferred first-line regimen is comprised of tenofovir disoproxil fumerate (TDF), lamivudine (3TC) or emtricitabine (FTC), and efavirenz (EFV)(40). Alternative regimens allow for zidovudine
(AZT) in place of TDF, and nevirapine (NVP) in place of EFV(40). For children
three years of age and up, the recommenced first-line includes EFV, 3TC, and the NRTI abacavir (ABC). Until recently, regimens in RLS often contained stavudine (d4T), however as a result of high toxicity associated with its use, d4T use is declining.
HIV Drug Resistance
The reverse transcriptase enzyme, responsible for converting viral RNA into cDNA (Figure 1), lacks a proof-reading capability resulting in, on average, one mutation per genome per replication cycle(42). A lack of proof reading
ability and frequent recombination events(43, 44), combined with the high
turn-over of virus particles, on average 109 -1011 virus particles daily(45, 46), results
in a multitude of different viruses within a population. This genetically diverse population of viruses is referred to as viral quasispecies(47), and can include
viral variants containing drug resistance mutations (DRMs)(48, 49).
Mutations conferring drug resistance are introduced in the viral genome at reverse transcription, and the proviral DNA containing these mutations is subsequently integrated into the cellular genome. Even when viral replication is suppressed by effective treatment, integrated proviral DNA is still present in cells. Virus variants produced from proviral DNA encoding resistance often have a reduced replication capacity and are out competed by wild-type virus. However, incomplete suppression of viral replication due to suboptimal drug pressure as a result of poor adherence, treatment interruption, insufficient plasma drug concentrations, or the use of inferior drugs or drug combinations, may result in a selective replicative advantage for viral variants with resistance to that drug. In such a situation, resistance virus can rapidly become the dominant virus, decreasing efficacy of the particular ART regimen and resulting in ARV treatment failure. As such, sub-optimal bioavailability of the ARV leads to the acquisition of drug resistance. In addition, individuals harbouring drug resistant virus may transmit this virus to previously uninfected individuals, referred to as transmitted drug resistance (TDR).
DRMs are specific to the drug class they affect. Reduced NRTI susceptibility is associated with specific mutations at 16 amino acid positions in RT(50).
Similarly, various mutations conferring reduced susceptibility to NNRTIs have been identified, as well as for PIs and inhibitors of the viral integrase enzyme.
1
Within and between drug classes, those mutations can have either additive, synergistic or antagonistic cross-reactivities, which makes interpretation of mutational patterns for clinical drug efficacy purposes a rather complex and specialised matter.
The WHO established a global HIV Drug Resistance Network (HIVResNet) in order to monitor the emergence of HIVDR. Countries in Africa that are currently involved include Burundi, Cameroon, Kenya, Malawi, Mozambique, Nigeria, South Africa, Swaziland, Zambia, and Zimbabwe. In the 2012 report, results of the WHO survey of acquired drug resistance obtained between 2006 and 2010 have shown that of the individuals failing therapy (VL >1000 copies/ml) at 12 months, 72% had DRMs (Figure 6). The most common mutation observed is the NRTI mutation M184V followed by several NNRTI mutations, such as K103N, Y181C and V106M(51). Due to the limited use
and later introduction of PI containing regimens, the occurrence of PI drug resistance is still low(51).
Figure 6. Prevalence of HIV drug resistance-associated mutations among people
experiencing treatment failure at 12 months. Mutations were defined using the 2009 WHO surveillance resistance mutation list. (Source: WHO Resistance Report 2012(51)).
According to the WHO resistance report for 2012, there has been an increase in TDR between 2004 and 2010 in RLS areas conducting HIVDR surveillance, either in published studies or as part of the WHO surveys(51).
Of note was the increase and prevalence of NNRTI resistance. A slightly increased prevalence of HIVDR was seen in countries with high national ART coverage(51), with the level of TDR being linked to the duration of national
roll-out programs(52, 53).
Treatment Monitoring
During the progression of HIV infection (Figure 7), an increase in viral replication generally precedes a decrease in CD4+ lymphocytes(54), which,
in the long-term, is then followed by the presentation of AIDS-defining illnesses. VL monitoring is an effective tool to monitor for the first signs of treatment failure. The role of CD4+ lymphocyte counts is to gauge the state of the immune system to determine whether there is a risk for infection by opportunistic infections.
Figure 7. A generalised graph of the progression of HIV infection. (Adapted from
1
In RRS, the standard of care for HIV treatment monitoring employs CD4+ counts and VL monitoring at entry into care and every three to six months thereafter. In addition to VL and CD4+ testing, resistance testing is often preformed prior to treatment initiation in order to ensure the most effective choice of therapy,as well as at virological failure in order to select an appropriate follow-up regimen(56).
By contrast, treatment monitoring in most RLS primarily involves clinical monitoring for stage 3 and 4 AIDS-defining illnesses and, if available, immunological monitoring using CD4+ counts(40). The inadequacies of CD4+
counts for determining treatment failure have been described on many occasions, either leading to unnecessary switching of regimens due to incorrectly diagnosed or presumed virological failure(57-61), or an accumulation
of drug resistance mutations due to extended exposure to a failing regimen(62).
Recently, the WHO added VL monitoring as a strong recommendation for diagnosis and confirmation of treatment failure in RLS(40), however its use is
limited due to costs and lack of laboratory infrastructure. Similarly, the use of HIVDR genotyping for individual patient management in RLS is limited to clinical research studies, and for pre-authorized private medical care, or at the patients own expense. Limited routine resistance testing is performed due to high cost, infrastructure requirements, and complexity of available assays.
Logistics of Treatment Monitoring
Commercial VL and HIVDR genotyping assays are performed from plasma, which require cold-chain maintenance and biohazardous containment. This limits their use in RLS to collection sites with appropriate sample processing facilities and cold-storage, which are also within easy reach of reference laboratories for subsequent testing. The use of dried blood spot samples (DBS) is becoming increasingly popular for VL monitoring and HIVDR genotyping in RLS. Numerous studies have been conducted to determine potential application of DBS in various assays, reviewed by Hamers et al. (2009)(63)
and Bertagnolio et al. (2010)(64). Although initial data suggests application is
feasible for VL and HIVDR genotyping, findings are inconsistent with respect to storage temperature and duration, and more detailed analysis on the effect of storage on sample degradation is necessary to ensure reliable results. Additionally, the sampling technique has numerous drawbacks: small sample volumes decrease analytical sensitivity, impaired nucleic acid integrity when stored in sub-optimal conditions, laborious nucleic acid isolation procedures limiting high throughput capacity, and potential interference of archived proviral DNA.
Treatment Challenges in RLS
The majority of HIV-infected individuals reside in Africa. Whilst access to treatment is increasing, there are still logistical hurdles to prevent HIVDR and maintain long-term treatment success. The largest risk factor associated with the development of HIVDR is adherence. The recent introduction of once-daily fixed dose cART greatly decreased pill burden and should improve patient adherence; however, adherence is still hindered by drug stock outs due to poor supply and procurement management, as well as limited supportive care, including VL and CD4 testing, resulting in extended exposure to failing regimens.
In addition to logistical hurdles, the vast array of subtypes found in Africa add an additional challenge to treatment and testing. Some resistance mutations are more prevalent in certain subtypes due to differences in the genetic make-up between subtypes thereby facilitating the emergence of some mutations over others. For example, studies have shown that the prevalence of K65R, conferring reduced susceptibility to d4T, ddI, ddC, 3TC, FTC, ABC, and TDF(50), is higher in nonsubtype-B clades(65-69). Similarly, a higher prevalence
of the V106M mutation over the V106A is observed in subtype C viruses, which is associated with high-level EFV resistance(70, 71). Whilst individual
patient management is currently not feasible in RLS due to costs, population-based surveillance for HIVDR mutations prevalence and subtype distribution
1
is necessary in order to make the most effective choice of ARV therapy for national guidelines.
Scope of This Thesis
Affordable Resistance Test for Africa: ARTA
Established in 2008, the ARTA project is a private-public consortium headed up by the PharmAccess Foundation, a non-profit organisation affiliated with the Center for Poverty-related Communicable Diseases of the Academic Medical Centre (AMC) in Amsterdam. The collaborators involved in the project include the University Medical Centre Utrecht (UMCU) in the Netherlands, the Centre de Recherche Public de la Santé in Luxembourg, the University of the Witwatersrand and Contract Laboratory Services in Johannesburg, South Africa, and Janssen Diagnostics BVBA (formerly Virco BVBA) in Belgium. The ARTA project aimed to develop more affordable HIV-1 treatment monitoring applications, including VL monitoring and HIVDR genotyping, which can be universally applied in RLS. The specific requirements are given in Box 1.
Box 1. Specific requirements for ARTA assays:
Simplified sample collection and transport (DBS) Optimized nucleic acid elution methodologies for DBS HIV subtype-independent assays nucleic acid amplification Pragmatic genotyping methods
Commercial VL assays provide precise measurement of viral RNA copies found in a sample, but generally require large equipment designed for high-throughput to ensure cost effectiveness, which is not always ideal for small- and medium-throughput facilities. However, 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. As such, a simple virological failure (VF) screening assay that is compatible for application with DBS was
developed, validated and transferred to three laboratories in Uganda with various levels of experience on application of molecular monitoring methods
(Chapter 2).
A recent review of ARV treatment programs in sub-Saharan Africa has shown that 94% of treatment is first-line based, and effective virological suppression is achieved for approximately 80% of these patients(72). Similarly, in the 2012
WHO survey of acquired drug resistance, 76% of patients achieved virological suppression at 12-months after initiation of a standard first-line regimen(51).
However, 20-24% of patients failing cART did not have DRM, and would have had unnecessary treatment switching without access to HIVDR genotyping results. Taking into consideration that the majority of HIV infected individuals receive first-line cART, a low cost HIVDR genotyping assay targeting the region of RT harbouring all major RT inhibitor resistance mutation positions was developed and applied clinically for HIVDR determination in first-line therapy failure (Chapter 3). Further, evaluation of the application of newly
developed VL and HIVDR genotyping assays in RLS is shown to demonstrate feasibility in a South African laboratory (Chapter 4).
For more in depth studies on first- and second-line drug resistance, an alternative to commercial PR-RT based HIVDR genotyping assays is described (Chapter 5), and is integrated application in RLS-related studies is
shown (Chapters 6, and 7). Lastly, the stability of DBS samples is important
to ensure reliable results using DBS sampling, and a further understanding of the effect of storage conditions On DBS sample integrity is explored
(Chapter 8). A final discussion (Chapter 9) concludes the relevance of the
described research.
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C
A
H
P
T
E
R
Susan C Aitken*, Aletta Kliphuis*, Michelle Bronze, Carole L. Wallis, Cissy Kityo, Sheila Balinda, Wendy Stevens, Nicole Spieker, Tulio de Oliveira, Tobias F. Rinke de Wit and Rob Schuurman.
*These authors contributed equally
of an affordable real-time
qualitative assay for
determining HIV-1 virological
failure in plasma and dried
blood spots
Abstract
V
irological failure (VF) has been identified as the earliest, most pre-dictive determinant of HIV-1 antiretroviral treatment (ART) failure. Due to high costs and complexity of virological monitoring, VF assays are rarely performed in resource-limited settings (RLS). Rather, ART failure is determined by clinical monitoring and to a large extent immunological monitoring. This paper describes the development and evaluation of a low-cost, dried blood spot (DBS)-compatible qualitative assay to deter-mine VF, in accordance with current WHO guideline recommendations for therapy-switching in RLS. The described assay is an internally-controlled qualitative real-time PCR targeting the conserved long terminal repeat do-main of HIV-1. This assay was applied with HIV-1 subtypes A-H and further evaluated on HIV-1 clinical plasma samples from South Africa (n=191) and Tanzania (n=42). Field evaluation was performed in Uganda using local clinical plasma samples (n=176). Furthermore, assay performance was evaluated for DBS. The described assay is able to identify VF for all major HIV-1 group-M subtypes with equal specificity, and lower detection limit of 1.00E+03 copies/ml for plasma and 5.00E+03 copies/ml for DBS. Com-parative testing yielded accurate VF determination for therapy-switching in 89%-96% of samples compared to gold standards. The assay is robust and flexible, allowing for “open platform” applications and producing com-parable results to commercial assays. Assay design enables application in laboratories that can accommodate real-time PCR equipment, allowing decentralization of testing to some extent. Compatibility with DBS extends access of sampling and thus access to this test to remote settings.2
IntroductionI
n 2010 the HIV-1 epidemic was estimated to include 34.0 million (range 31.6-35.2 million) infected adults and children across the globe. An alarm-ing 67.4% (n= 22.9 million) of the total global infections are people residalarm-ing in Sub-Saharan Africa. As a result of antiretroviral therapy (ART) scale-up initiatives, 6.65 million of infected individuals requiring treatment were receiv-ing it in Sub-Saharan Africa by the end of 2010(1). However, particularly inre-source-limited settings (RLS), effective treatment faces challenges, which in-clude failing supply chains resulting in drug stock-outs, drug toxicity of “older generation” 1st and 2nd line drugs, failure of patient adherence, drug-drug
in-teractions, lack of qualified healthcare staff or failing adherence support, etc. As a result HIV-1 can develop drug resistance to ART, leading to virological failure (VF) and subsequently ART failure. A recent report has shown that the prevalence of pre-ART HIV-1 drug resistance in 13 sites in various countries in Sub-Saharan Africa is 5.6%, ranging from 1.1% in South Africa to 12.3% in Uganda(2). Recent scientific findings have led to the consideration of
“Treat-ment as Prevention”, which according to the most intensive “Test and Treat” scenario could ultimately increase the number of HIV patients qualifying for ART to 32 million(3). With rapidly increasing numbers of HIV patients on ART
in RLS with weak health systems, the risk of further increase of HIV-1 drug resistance is imminent.
The success of increased access to ART in RLS has largely been due to massive donor funding and important reduction of costs of selected first-line drugs. However, reduced susceptibility to these first-first-line drugs and the consequent switching to second-line would at least partly undo early ART successes and result in higher expenditures and increasing numbers of pa-tients on failing regimens with no options for effective second-line or salvage therapies(4, 5).
As per the definition of the WHO, VF is a repeated viral load ≥5.00E+03 RNA copies/ml in an individual taking ART for at least four to six months(6).
Timely detection of VF by VL testing, which is routine in industrialized coun-tries(7), is necessary to prevent accumulation of HIV drug resistance(8), or to
identify poor adherence to the treatment. However, in RLS, high costs and technical complexity limit the possibility of VL monitoring and treatment fail-ure is primarily determined using clinical monitoring for stage three and four AIDS-defining illnesses and, if available, immunological monitoring using CD4 counts(6). The inadequacies of CD4 for determining true treatment
fail-ure have been described on many occasions (9-12). The clinical-immunological
monitoring approach results in individuals being left on sub-optimal regimens for an extended period of time with the risk of accumulating drug resistance mutations or unnecessary switching to second-line therapy based on non-VL supported decisions(4). Both scenarios limit future treatment options and
increased costs associated with second-line therapy(5).
The current paper addresses the challenge of determination of VF in RLS by taking several premises into consideration that reflect the actual public health situation in these settings. First of all, the standpoint was taken that determination of an exact VL is not required to determine ART failure and therefore a less complex, and thus less expensive, assay that classifies a sample as either above or below a treatment success threshold would suf-fice. Secondly, in order to implement the WHO recommendations of task shifting and decentralization of ART to remote settings, the consequence would be that complex procedures, including drawing blood, isolation and storage of plasma, cold chain shipments to qualified labs for VL testing, should be avoided. Rather, VF should be detectable on dried blood spots (DBS), a sampling alternative that is inexpensive, easy to collect and trans-port, and has proven application for VL testing(13, 14). Thirdly, given the fact
that for accurate detection of VF a nucleic acid amplification step remains necessary and taking into consideration the realities of contamination risks in remote labs, it was decided to concentrate on a real-time PCR approach. This allows for VF determination in a closed system and with equipment that is continuously evolving, regularly reducing in price, and being adapted to
2
local circumstances through battery and solar energy applications. Finally, it was considered essential that the protocol for VF testing should be generic, “open platform”, applicable on a wide array of real-time PCR instruments in various African settings, and freely available in the public domain.
With the above assumptions in mind, the Affordable Resistance Test for Af-rica (ARTA) consortium was established, consisting of a unique combina-tion of academia, industry and non-government organizacombina-tions both in Africa and Europe. Here we report on the results of ARTA research to develop a real-time PCR assay that can be used as a screening tool to determine VF in RLS. This “virological failure assay” (VFA) can be readily applied in basic laboratories, using either plasma or DBS as the sample input. The VFA is applicable for all major HIV-1 group-M subtypes, and is specifically designed to identify VF as defined by the WHO as a VL of ≥5.00E+03 copies/ml(6). Materials and Methods
Samples
HIV-1 Subtype Reference panel
A panel of virus isolates consisting of HIV-1 subtypes A through H (Table 1) was obtained from BBI (BBI Biotech Research Laboratories Inc., Gaithers-burg, USA) for assay optimization and evaluation at the University Medical Centre in Utrecht (UMCU), The Netherlands. Serial dilutions were prepared from these stocks using HIV-1 negative human plasma. These dilutions were also used to spike HIV-1 negative whole blood for DBS preparation.
Clinical samples
Clinical plasma samples from HIV-1 infected individuals from several African sites were included for further evaluation at the UMCU, the Netherlands. Samples were selected to include several subtypes with a variety of VL in ac-cepted ranges for subsequent analysis. Samples from South Africa (n=191) were plasma samples sent for routine VL testing, performed on the
CO-BAS®AmpliPrep/COBAS®TaqMan®System v2 (Roche, Penzberg, Germa-ny), and represented HIV-1 subtype-C with a VL range of 1.30E+03-3.00E+06 (median 5.50E+04) copies/ml. Samples from Tanzania (n=42) were part of an ongoing prevention-of-mother-to-child-transmission (PMTCT) study(15),
where VL was determined using the COBAS® Amplicor HIV-1 Monitor test v1.5 (Roche). Samples included subtypes A (n=23), C (n=10), and D (n=6), and three samples with undetermined subtype, with a VL range of 6.65E+02-3.07E+05 (median 2.67E+04) copies/ml.
Table 1. HIV-1 isolated from the BBI subtype reference panel
Subtype Strain Country of Origin Accession Number
A UG275 Uganda AB485632
B BK132 Thailand AY173951
C ZB18 Zambia AB485641
D SE365 Senegal AB485648
CRF01_AE CM240 Thailand AF067154
F BZ126 Brazil AY173957
G BCFDIOUM Zaire AB485661
H BCPKITA Zaire AB485665
In addition, as part of a technology transfer program, the described assay was applied in three Joint Clinical Research Centre (JCRC) laboratory sites in Uganda, where retrospective plasma samples collected from HIV-1 posi-tive individuals as part of the PASER program were included(16). These
sam-ples represented baseline and follow-up clinical samsam-ples at yearly intervals after therapy initiation. For these samples, routine VL had been performed in Uganda using the COBAS®AmpliPrep/COBAS®TaqMan®System v2(Ro-che). A total of 176 plasma samples were included comprising of subtypes A (n=89), D (n=64), and 23 with an unknown subtype, with a VL range of 1.00E+02-1.00E+06 (median 1.00E+04) copies/ml. Twenty-five confirmed HIV-1 negative plasma samples were included for assay specificity control.
2
To investigate the application of the assay with DBS samples, DBS were prepared from EDTA collected whole blood for participants of the PASER program(16). The same blood sample was then centrifuged and the plasma
was removed for analysis. These samples are subsequently referred to as paired plasma and DBS samples. A total of 82 paired samples were tested in Uganda, with a VL range of 4.40E+01-7.18E+06 (median 2.61E+03) cop-ies/ml. DBS samples were stored at -70°C for 270-515 (median 485) days (n=31), -20°C for 45-112 (median 82) days (n=21), or room temperature for 2-192 (median 126) days (n=30).
Internal Control
An internal control (IC) was added to each clinical sample at a fixed amount of ten percent of the elution volume at the start of nucleic acid isolation. The IC comprised of the non-human RNA virus Encephalomyocarditis virus (EMC) and was prepared at the UMCU, the Netherlands, in batches of sin-gle-use aliquots and stored at -80°C until use.
VFA
Nucleic Acid Isolation, Plasma
At the UMCU, the Netherlands, viral nucleic acid (NA) isolation was per-formed using NucliSENS magnetic extraction reagents in combination with the MINIMAG (BioMérieux, Boxtel, The Netherlands). For each sample, an input of 100μl plasma was used, or two DBS of 50μl each, and 2.5μl IC. Pos-itive and negative controls were included in each run. Upon completion of the isolation procedure, purified nucleic acids were eluted in 25μl elution buffer. In Uganda, NA isolation was performed using the QIAamp RNA kit (Qia-gen GmbH, Germany), as per manufactures instructions. Input was 100μl of plasma, or two 50 μl DBS, and 5μl IC. Isolated NA was eluted in 50μl elution buffer.
Upon completion of both isolation procedures, the eluates were used imme-diately for reverse transcription (RT), and remaining nucleic acids stored at -20°C.
Nucleic Acid Isolation, DBS
At both sites, a pre-incubation step for DBS was performed. DBS samples where excised by hand using scissors, which were decontaminated between samples with 70% ethanol. For the Nuclisens method, DBS were placed in the provided 2ml lysis buffer (BioMérieux) in a 9ml tube. For the QIAamp RNA method (Qiagen GmbH), DBS were placed in 700ul of the provided Buffer AVL lysis buffer that was aliquoted in 2ml Eppendorf tubes for use. For both methods, samples were incubated at room temperature with gentle rotation for 30 minutes, after which filters were removed and NA isolated as per the plasma samples.
Reverse Transcription
Purified NA, containing both HIV-1 RNA and IC RNA, was reverse transcribed using the TaqMan® Real-time PCR system Random Hexamers RT kit (Life Technologies, Foster City, CA) according to manufactures instructions. An in-put of 10μl NA isolate was used in a final reaction volume of 25μl. Reactions were carried out according to the following conditions: 25C° for 10 minutes, 48°C for 30 minutes and 95°C for 5 minutes. Generated cDNA was used immediately for real-time PCR or stored at 4°C.
Real-time PCR
HIV-1 and IC cDNA fragments were amplified in multiplex format. A 25μl real-time PCR reaction contained 12.5μl universal TaqMan® Master Mix (Life Technologies), 10μl cDNA, 300nM primer EMC-forward, 900nM primer
EMC-reverse, 100nM probe EMC-VIC, 300nM forward primer LTR S4, a
mix-ture of 600nM HIV-LTR revere primers 3’UNI-KS-6 and 3’UNI-KS-6-AG, and 100nM MGB probe HIV-LTR-FAM (Table 2). The assay was performed at the UMCU, the Netherlands, using an Applied Biosystem 7500 Real-Time PCR
2
System (Life Technologies), and in Uganda using a MiniOpticonTM Real-Time
PCR Detection System (BioRad, Hercules, CA). Both systems included a temperature profile allowing for dUTP/UNG decontamination, namely 50°C for 2 minutes; 95°C for 10 minutes; 45 cycles of 95°C for 15 seconds and 60°C for 1 minute. In order to enable run-to-run comparison, a fixed thresh-old was established for both systems (data not shown).
Assay Controls
Positive and negative controls were included in each run. Appropriate per-formance of the run was judged based on the results of these controls. The positive control consisted of a plasma sample spiked with a fixed concentra-tion of 2.50E+04 copies/ml HIV-1, the Ct value acceptance range of which was determined for each real-time system. For this evaluation the positive control range was set at Ct 29-32. The IC was used to monitor for inhibition of each individual sample. As with the positive control, the Ct value acceptance range of the IC was determined for each real-time system. For this evalua-tion the positive control range was set at Ct 30-33. Three negative controls were included, an isolation negative that consisted of HIV-1 negative human plasma and IC, and negative RT and PCR controls which consisted of nucle-ase-free water and no IC. The result obtained for a sample was considered valid when the positive and IC controls were within their predetermined rang-es, and the negative controls were below detection.
Data Analysis
Assay Range
A five-fold serial dilution series of viral RNA for plasma and DBS for all panel subtypes (Table 1) was used to assess dynamic range, level of detection (LOD), and inter- and intra-assay reproducibility. For the ABI7500 (Life Tech-nologies) the serial dilution series ranged from 5.00E+06-3.20E+02 copies/ ml, and for the MiniOpticon (BioRad) from 1.00E+06-1.6E+03 copies/ml. Lin-earity was determined and reported as a R2 value and slope gradient.
Table 2.
Primer and probe sequences for the HIV
-1 V
irological Failure screening
Assay . Primer/Probe Sequence Function Target EMC-forward 5’-TGACCACGCCACCGC-3’ Forward primer EMC EMC-reverse 5’-T AAAGA TTTCCCTTGCCCCG-3’ Reverse primer EMC EMC-VIC 5’-TGTGAGCCAGTCGTGA TTGTGCTCC-3’ Probe EMC HIV -L TR S4 5’-AAGCCTCAA TAAAGCTTGCCTTGA-3’ Forward primer HXB2 nt520-543 3’UNI-KS-6 5’-GAGGGA TCTCT AGTT ACCAGAGTCACA-3’ Reverse primer HXB2 nt574-600 3’UNI-KS-6-AG 5’-GAGGGA TCTCT AGTT ACCAGAGTCCT A-3’ HIV -L TR-F AM 5’-T AGTGTGTGCCCGTCTG-3’ MGB probe HXB2 nt554-570
EMC: Encephalomyocarditis virus (internal control): L
TR: long terminal repeat region of HIV
-1; HXB2: HIV
