j o ur nal ho me pag e: www.elsev ier .com/ locate / jv i romet
evelopment of a cost-effective assay for genotyping of HIV-1 non-Bubtype for drug resistance
alanee Ammaranonda,b,∗, Sayompoo Sanguansittianantb, Paul A. Rajuc,hilip Cunninghamd, Navin Horthongkhame
Department of Transfusion Medicine, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok, ThailandInnovation Center for Research and Development of Medical Diagnostic Technology Project, Department of Transfusion Medicine, Faculty of Allied Healthciences, Chulalongkorn University, Bangkok, ThailandNational Engineering Research Center for Miniaturized Detection System, Northwest University, Xi’an, ChinaSt Vincent’s Hospital Sydney Limited, Sydney, AustraliaDepartment of Microbiology, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand
rticle history:eceived 28 August 2013eceived in revised form 4 January 2014ccepted 10 January 2014vailable online 22 January 2014
eywords:enotyperug resistanceIV-1
a b s t r a c t
Highly Active Antiretroviral Therapy (HAART) is the most effective way to control HIV-1 replication ininfected patients. Prior to the start of therapy, genotyping of HIV-1 for mutations that confer resistanceto potential drug candidates is crucial for it allows formulating an effective regimen. Ineffective drugsare excluded and potentially effective ones are included. A number of diagnostic kits are commerciallyavailable for this purpose but are tailored for HIV-1 subtype-B, a strain chiefly found in AIDS patientsof Europe and America. However, AIDS patients of South-East Asia including Thailand are predominantinfected with HIV-1 subtype non-B. In this study, an inexpensive assay was developed that genotypesHIV-1 non-B for drug resistance and tested it on 99 Thai AIDS patients. Results showed that 98 wereinfected with HIV-1 subtype non-B (or CRF01 AE) and one with subtype-B. Within the HIV-1 polymerase(pol), reverse transcriptase (RT) gene, the assay identified 18 codon mutations associated with resis-tance to Nucleoside/Nucleotide Reverse Transcriptase Inhibitors (NRTIs) and 17 Non-Nucleoside ReverseTranscriptase Inhibitors (NNRTIs). Employing a commercially available kit, parallel genotyping of patient
samples confirmed results providing validation of the assay. This method approximately costs 100 USdollars compared to $300 for a commercially available test. In Thailand, the burden of cost for treatingHIV-infections is high not only for the average citizen but the country’s health care systems. Thereforethe low cost and yet effective genotyping test for HIV-1 subtype non-B is a practical and viable solution
platfo
to expensive genotyping
. Introduction
Thailand’s Department of Disease Control & Ministry of Publicealth estimates that between 1984 and 2012 there were 276,947eople with AIDS and more than 1.2 million were infected with
IV (Bureau of Epidemiology, Thailand). Currently, Thailand hasne of the highest HIV-1 infection rates among Asian countrieshich financially burdens government and private health care
Abbreviations: HAART, Highly Active Antiretroviral Therapy; NRTIs, Nucleo-ide/Nucleotide Reverse Transcriptase Inhibitors; NNRTIs, Non-Nucleoside Reverseranscriptase Inhibitors.∗ Corresponding author at: Department of Transfusion Medicine, Faculty of Alliedealth Sciences, Chulalongkorn University, 154 Chulapat1 Building, Rama I Road,athumwan, Bangkok 10330, Thailand. Tel.: +66 2 218 1084x321/683 603 4334;ax: +66 2 218 1083.
systems. The National Health Security Office (NHSO) and NationalHealth Expenditure for HIV infection and AIDS reported that in2010 and 2011 approximately 200 million US dollars was spent onAIDS related health care measures which include diagnostic test-ing and providing effective medication (National Health SecurityOffice, Thailand).
Controlling the spread of HIV-1 among the masses is achievedthrough implementing safe health practices and marginalizing theviral reservoir in infected patients with effective therapy. It hasbeen well established that antiretroviral therapies curb viral loadand maintain adequate CD4+ T lymphocyte counts (Safren et al.,2005; Chitra et al., 2009; Manosuthi et al., 2009; Azzoni et al.,2011; Torti et al., 2011). Active replication in an infected patientcan produce anywhere from 1010 to 1011 virions per day and
given the high error rate of HIV-1 reverse transcriptase, there isa high probability that viral mutants resistant to a given drug/classof drugs arise (Preston et al., 1988; Hu and Temin, 1990; Holtz
nd Mansky, 2013). Furthermore drug resisting mutants can alsoesult from genetic recombination mechanisms between two dis-inct proviral HIV-1 genomes within an infected cell (Hu and Temin,990). For these reasons a regiment of two or more antiretrovi-als (HAART) has proven to an effective course for treating HIV-1nfections. Current HAART formulations encompass a spectrumf compounds that vary in their inhibition mechanism of viraleplication. These include Nucleoside/Nucleotide Reverse Tran-criptase Inhibitors (NRTIs), Non-Nucleoside Reverse Transcriptasenhibitors (NNRTIs), Protease Inhibitors, Fusion Inhibitors, and Inte-rase Inhibitors (Ammaranond and Sanguansittianan, 2012).
Identifying mutations in a patient’s viral strain that can con-er resistance to an antiviral or class of antivirals is the basis of
genotype based test. Existing commercial genotype kits assesshe drug resistance of HIV-1 subtype B, a strain found in patients ofurope and America (UNAIDS). In Southeast Asia including Thailandatients are mostly infected with the recombinant form AE oron-B subtype (Manosuthi et al., 2013; Raymond et al., 2013;anguansittianant et al., 2013). Some studies have shown that com-ercial HIV-1 subtype B kits work well for various HIV-1 subtypes
ut with non-B subtypes results have not fared well (Fontaine et al.,001; Ariyoshi et al., 2003; Beddows et al., 2003; Eshleman et al.,004). In Thailand, a typical genotypic assessment for HIV-1 drugesistance costs a patient between 300 and 500 US dollars (Nationalealth Security Office, Thailand), which is expensive for a citizenith average income. This combined with the lack of a sensitive
est of HIV-1 non-B subtype has provided with the impetus for thisork.
. Materials and methods
.1. Sample preparation and extraction of HIV-1 RNA
Peripheral blood from HIV-1 patients was provided by Theealth Science Service Unit, HIV Laboratory, Faculty of Allied Healthciences, Chulalongkorn University. Immediately upon collection<6 h), blood in vacutainers (w/anticoagulant ethylenediaminete-raacetic acid, EDTA) was centrifuged at 1500 rpm for 10 min, 1 mlf plasma was removed for HIV RNA quantification with COBAS®
aqman® HIV-1 test (Roche Molecular Diagnostics, Switzerland)nd 1 ml of plasma was stored in sterile cryovials at −70 ◦C for RNAxtraction.
Cryovials with frozen plasma were rapidly thawed and 650 �l oflasma with over 2000 copies/ml of viral load, was extracted withOBAS® AmpliPrep Total Nucleic Acid Isolation kit (Roche Molecu-
ar Diagnostics) and genotyped by in-house test in parallel with theommercial kit Trugene HIV Genotyping test (Siemens Healthcareiagnostics, Australia).
.2. Reverse transcription of patient HIV-1 RNA into cDNA
Fig. 1 flow charts the strategy of in-house assay. To confine theenotype test to HIV-1 non-B, 4 primers were designed to amplifyelevant regions of the polymerase (pol) gene of CM240, an HIV-1train found predominately in Thailand’s AIDS patients (GenBanko. AF447851.1):
HIV-1 RNA in extracted samples was reverse transcribed witholoney Murine Leukemia Virus reverse transcriptase (MMLV RT,
romega, USA). The reaction was carried out by placing 10 �l of RNA
ical Methods 199 (2014) 102–107 103
into PCR tubes, heated to 70 ◦C (∼5 min) to denature its secondarystructure, cooled (on ice for ∼5 min), mixed with 15 �l of RT-PCRmaster mix and thermocycled (GeneAmp, USA) for one round ofreverse transcription; 42 ◦C for 45 min, 95 ◦C for 5 min, 4 ◦C hold andall ramp times were 1 ◦C/s. Per-reaction, the master mix consistedof 5.0 �l of 5xMMLV RT buffer, 2.0 �l of 25 mM MgCl2, 2.0 �l of2.5 nM dNTP, 1.0 �l of 10 �mole/�l RT-PCR-end primers, 4.25 �l ofdistilled water, 0.5 �l of 200 U/�l M-MLV RT enzyme and 0.25 �l of40 U/�l RNase-Inhibitor.
2.3. Amplification of transcribed cDNA
HIV viral genome was amplified on pol. For standard PCRmethod, 10 �l of the transcribed cDNA was transferred into PCRtubes containing 40 �l of master mix and thermocycled for onecycle at 94 ◦C for 4 min, 35 cycles at (94 ◦C for 30 s, 55 ◦C for 30 s,72 ◦C for 2 min) and one cycle at 72 ◦C for 7 min with a 4 ◦C hold. Allramp times were at 1 ◦C/s. Master mix contained per reaction: 5.0 �lof 10xTaq buffer, 2.0 �l of 25 mM MgCl2, 4.0 �l of 2.5 mM dNTP,1.5 �l of 10 �mole/�l RT-PCR-end primers, 1.5 �l of 10 �mole/�l ofPA3 primers, 25.5 �l of distilled water, and 0.5 �l of 5 U/�l of TaqDNA polymerase (Thermo Fisher Scientific, EU).
For nested PCR technique, 5 �l of PCR product from the aboveamplification in PCR tubes was mixed with 45 �l of master mixand thermocycled with the same settings of standard PCR methodas above. Nested PCR master mix contained per reaction: 5.0 �Lof 10xTaq buffer, 2.0 �l of 25 mM MgCl2, 4.0 �l of 2.5 mM dNTP,1.5 �l of 10 �mole/�l RT-PCR-start primers, 1.5 �l of 10 �mole/�lPA1 primers, 30.5 �l of distilled water and 0.5 �l of 5 U/�l TaqDNA polymerase (Thermo Fisher Scientific, EU). The expected sizeof PCR product of 904 bp was confirmed by 0.7% agarose gelelectrophoresis/UV lamp detection with 100 bp DNA markers (Fer-mentas, Thermo Fisher Scientific, EU). The final PCR product waspurified by HiYieldTM Gel/PCR Fragments Extraction Kit (RBCBio-science, Taiwan).
2.4. Sequencing of the amplified cDNA pol-RT gene
The amplified cDNA was sequenced employing 6 primers thatspecifically tailored to target expected mutations within the HIV-1reverse transcriptase region. The primers were of 20–30 bases longproviding ideal melting temperature and annealing times for stan-dard PCR reactions. These primers (listed below) were employedby 1st BASE DNA Sequencing Services (Malaysia) to sequence theHIV-1 pol-RT gene extracted and amplified from patient samples.
For sequencing HIV-1 RNA samples with Trugene HIV Genotyp-ing Kit, standard operating procedures of the vendor were closelyfollowed. In brief, cDNA of patient samples were PCR amplifiedfor HIV-1 pol genes (297 and 1680 nucleotides in length), ven-dor primers targeted protease and reverse transcriptase regions.The amplified fragments were sequenced by a “CLIP” reaction that
employed fluorescently labeled forward and reverse primers. Uponcompleting of the reaction fluorescent fragments were detectedby polyacrylamide gel electrophoresis using Long-Read TowerSequencer (Siemens Healthcare Diagnostics, Australia).
104 P. Ammaranond et al. / Journal of Virological Methods 199 (2014) 102–107
Fig. 1. Schematic illustration of in-house genotyping assay strategy. Reverse transcribed patient HIV-1 cDNA was methodically PCR-amplified to obtain RT fragments wepredicted-by online database searches-to contain drug resistant mutations. PCR primers PA3/RT-PCR-end generated a pol gene template from which primers RT-PCR-start/PA1a tweena le. RelI
2r
KbSsbAtNdfWt
3
3
rtows2srr
2004; Ceccherini-Silberstein et al., 2005; Menendez-Arias et al.,2006; Sanguansittianant et al., 2013). The remaining mutationsdetected are probably of viral transcriptional error. In the RT gene,
Table 1General characteristics of the sample population.
VL <50,000 copies/ml VL >50,000 copies/ml
Sample size 49 50Gender
• Male 19 18• Female 20 19• Unda 10 13
Subtype• CRF01 AE (Non-B) 49 49• B 0 1
VLb median (range) 12,400 (2030–42,400) 173,500 (50,900–2,160,000)
mplified th RT gene. Within the RT gene, primers RT41/RT74 amplified the region bend RT181/RT219 from position 181 to 219. Spatial distances are not drawn to scaNT = Integrase.
.5. Alignment of DNA sequences and data mining for drugesistance
With results obtained with the Trugene HIV Genotypingit, the forward and reverse CLIP DNA sequences are com-ined and compared to the HIV-1LAV-1 with the OpenGeneystem software (Siemens Healthcare Diagnostics, Australia). DNAequences obtained with both methods were edited and assem-led using a reference sequence on the DNA Base Sequencessembler (http://www.dnabaser.com). Data was mined for muta-
ions that can potentially give resistance to antivirals withinRTIs and NNRTIs bandwidth on the Stanford HIV drug resistanceatabase (http://hivdb.stanford.edu/) or Trugene HIV-1 Moduleor GuideLinesTM Rules. Statistical comparisons were made with
ilcoxon Matched Pairs Signed-Ranks Test (Statistics Package forhe Social Sciences, SPSS).
. Results
.1. General characteristics of the sample population
Table 1 summarizes the nature of samples genotyped for drugesistance. Out of 99 patients and on a per milliliter basis of plasmaested, 49 had an HIV-1 viral load of <50,000 copies with a medianf 12,400 copies, 50 samples had >50,000 copies where the medianas 173,500 copies. The median viral load for the entire sample
et (n = 99) was 46,650 copies/ml with a range between 2030 and
,160,000 copies/ml. Analysis of DNA sequence of the RT regionhowed that one sample was of HIV-1 subtype-B whereas theemaining 98 samples were CRF01 AE or subtype non-B. Theseesults were confirmed with the Trugene HIV Genotyping Kit.
amino acid (or codon) position 41–74, primers RT100/RT108 from position 100–108evant Abbreviations: pol = Polymerase, RT = Reverse Transcriptase, PR = Proteinase and
3.2. Identifying drug resistance-associated mutation codons inthe pol-RT gene
Fig. 2 summarizes drug resistance-associated codons mutationsidentified by the assay. A total of 18 for NRTI and 17 for NNRTI.M184 (NRTI) was the most prevalent found in 69 of the 99 samplesgenotyped whereas H208 was least, occurring in only one sample.As for NNRTI resistance, Y181 was dominant, found in 45 sampleswhereas mutations F227, M230 and K238 were rare being presentin only one sample. It is well known that D67 is a deletion muta-tion whereas T69 is an insertion (Imamichi et al., 2001; Miller et al.,
Blood collected from HIV-1 infected patients were from of Bangkok (Thailand) andsurrounding vicinities. Patients who wished to not declare their sexual identity arecategorized as undeclared. HIV-1 was subtyped from its cDNA sequence.
P. Ammaranond et al. / Journal of Virological Methods 199 (2014) 102–107 105
Fig. 2. In-house assay detection of NRTI and NNRTI resistance-associated codon mutations. A mutation frequency here is defined as the percentage of samples positive for ag resistd
das2Wtt(e
watcchdamai1(Ph
oifmatSGw
roc1rE
Sanguansittianant et al., 2013). Phenotypic drug resistance testsare laborious, time consuming and more expensive than genotypictests and thus are not preferred for resource limited countries.
Table 2Mining for resistance-associated mutations to a specific antiviral on separatedatabases.
iven drug resistance-associated codon-data mining utilized the Stanford HIV drugetected (zero mutation frequency).
eletions between codon positions 67–70 occur with thymidinenalog associated mutations (TAMs) or Q151M complex, they areimilar but have weaker effects than a T69 insertion (Kisic et al.,008, 2011; Saravanan et al., 2012; Sanguansittianant et al., 2013).hen present with one or more TAMs at codons 41, 210, or 215,
he 69 insertion mutation is associated with resistance to all NRTIshat are currently approved by the Food and Drug AdministrationUSA) (Miller et al., 2004; von Wyl et al., 2010; Sanguansittianantt al., 2013).
To validate the assay, the same sample set was genotypedith the commercially available Trugene HIV-1 genotyping kit
nd mined for NRTI and NNRTI resistance-associated codon muta-ions. These results are shown in Fig. 3. The frequency of mutationommonly identified by both assays which were presented as aorrelation plot. A total of 35 mutations identified by both methodsave a 99.5% correlation thus validating the assay. Despite this highegree of correlation some minor differences have been noted; thisssay was able to better detect the frequencies of NNRTI-resistanceutations at amino acid (or codon) positions 90 (V90), 138 (E138),
nd 227 (F227), while the commercial kit was better at identify-ng mutations at positions 101 (K101), 103 (K103), 108 (V108),81 (Y181), 190 (G190), 221 (H221), 230 (M230) and 238 (K238)Ceccherini-Silberstein et al., 2005; Sanguansittianant et al., 2013).osition 318 (Y318) was not detected by the assay. This mutationas not been observed for first line drug regimen in the country.
The Trugene HIV-1 genotyping kit is calibrated to utilize itswn database (Trugene HIV-1 Module for GuideLinesTM Rules) fordentifying drug resistant mutations which relied on the Stan-ord HIV drug resistance database. When cross comparison was
ade if differences exist using the alternate database for eitherssay, only minor differences was found. One was the detection ofhe E44D/E mixture mutation (Ceccherini-Silberstein et al., 2005;anguansittianant et al., 2013). The Trugene HIV-1 Module foruideLinesTM Rules is limited in detecting this amino acid deletionhereas the Stanford HIV drug resistance database could.
However, this comparison was expanded in identifying drugesistance to a specific antiretroviral. Genotypic sequencesbtained by either assay were mined for resistance to eleven
ommon HIV-1 antiretroviral drugs on both the Trugene HIV-
Module for GuideLinesTM Rules and the Stanford HIV drugesistance database. These results are summarized in Table 2.mploying the Wilcoxon Matched Pairs Signed-Ranks Test, there
ance database. Mutation Y318 was not targeted by our primer design and thus not
were significant differences between the two assays (p < 0.05)for drugs Abacavir (ABC), Didanosine (DDI), and Efavirenz (EFV)when utilizing Trugene database. There were no differences for theother remaining drugs were noted. When utilizing the Stanforddatabase, no significant differences (p < 0.05) existed between thetwo assays for any one of the eleven drugs.
4. Discussion
The monitoring of CD4+ T lymphocyte count, HIV-1 RNA viralload and HIV drug resistance evaluates the infection status of apatient so-as-to determine whether antiretroviral therapy such asHAART should be prescribed (Torti et al., 2011; Kassler and Sticht,2013). In Thailand, the national guidelines for antiretroviral therapyfor HIV-1 infected adults and adolescents is a CD4+ T lymphocytecount <350 cells/mm3 (Sungkanuparph et al., 2010). Assessing apatient’s HIV-1 strain to drug resistance through either pheno-typic or genotypic drug resistance tests help formulate effectiveHAART medications (Ammaranond and Sanguansittianan, 2012;
p-Values were calculated using the Wilcoxon Matched Pairs Signed-Ranks Test. Boldfont = NNRTI drugs. Plain font = NRTI drugs.
106 P. Ammaranond et al. / Journal of Virological Methods 199 (2014) 102–107
Fig. 3. Validation of In-house assay with TRUEGENE HIV-1 genotyping kit. As in Fig. 2, a mutation frequency is the percentage of samples positive for a given drug resistance-associated codon. Results from TRUEGENE HIV-1 genotyping kit employed TRUGENE HIV-1 Module for GuideLinesTM Rules whereas In-house assay utilized the Stanford HIVd e arrav rgets
HifIcttCgddd
psawgctfig
nitsbTeaom
rug resistance database. In the figure insert, data points closely knit for labeling aralues. Mutation Y318 was identified by TRUEGENE kit in 2 samples for the assay ta
owever, genotypic drug resistance tests are still expensive accord-ng to the Comptroller General’s Department (Thailand). Medicalees for genotypic drug resistance assay cost ∼300–500 US dollars.n this study, a cost effective test was developed that specifi-ally genotypes of HIV-1 subtype non-B for drug resistance. Theest relies on the novel PCR primer design, where the pol geneemplate is generated with primers specific to HIV-1 viral strainM240 (a non-B subtype). This is followed by amplification of RTene segments with primers targeting known mutated codons forrug resistance. Gene segments were sequenced and mined forrug resistance-associated codons on Stanford HIV drug resistanceatabase.
Implementing the in-house test on 99 HIV-1 infected Thaiatients showed that 98 were positive for HIV-1 subtype non-B (A/Eubtype) strain. The assay successfully identified codon mutationsssociated with resistance to NRTIs and NNRTIs. In comparisonith the commercially available and established Trugene HIV-1
enotyping kit, NRTI and NNRTI mutation frequencies had ∼99%orrelation thereby validating the assay. Comparing this assay onwo different HIV drug resistance databases, Trugene HIV-1 Moduleor GuideLinesTM Rules and Stanford HIV drug resistance databases,t was found no major differences further solidifying the assay asenuine.
Besides being tailored for HIV-1 subtype non-B, this assay doesot require whole gene sequencing like TRUEGENE HIV Genotyp-
ng Kit and other genotyping assays. Only gene regions containingargeted antiretroviral resistance-associated mutation codons areequenced for subsequent bioinformatics. The biggest differenceetween genotyping method and commercial kits is the cost. Arugene HIV-1 Genotyping kit costs ∼300 US dollars/test. How-
ver, this assay would cost ∼100 US dollars/test which includesll the necessary elements (i.e., reagents, sequencing costs). Uponptimization it would be expected the cost to be even lower. Thisethod is cost-efficient, flexible, and easy to perform for drug
nged in increasing (bottom to top) order of mutation frequencies of In-house assay its location in the RT gene.
resistance testing. This might be an alternative way for investiga-tion of drug resistance mutation for HIV-1 non-B subtype.
Ethical approval
The study was approved by the Ethics Review Committeefor Research Involving Human Research Subjects, Health ScienceGroup, Chulalongkorn University-COA No. 080/2555.
Acknowledgement
The Ratchadaphiseksomphot Endowment Fund of Chula-longkorn University (RES560530214-HR).
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