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Genotype and antiretroviral drug resistance of human
immunodeficiency virus-1 in Saudi Arabia
Ghazi A. Jamjoom, PhD, FRCPath, Esam I. Azhar, MSc, PhD, Tariq A. Madani, MBBS, FRCP (C), Salwa I. Hindawi, MBBS, FRCPath,
Hanaa A. Bakhsh, MBBS, Arab Board (Pediatrics), Ghazi A. Damanhouri, MBBS, FRCPath.
ABSTRACT
monitoring. Samples were analyzed at the Special Infectious Agent Unit, King Fahd Medical Research )HIV-1( بستكلما ةعانلما صقن سوريف ةمواقم ليلتح :فادهلأا
Center of King Abdulaziz University, Jeddah, Kingdom ىضرلما ىدل سوريفلل ينيلجا عونتلا ديدتحو ةيرقهقلا تاسوريفلا تاداضلم of Saudi Arabia from August 2004 to June 2009. The لوب ينج نم ةمخضم ةقطنلم ينيلجا لسلستلا ةنياعبم كلذو ينيدوعسلا Viroseq2.5® kit (Celera/Abbott) was used with ABI Prism 3100 sequencer. All patients were Saudi nationals and were on antiretroviral therapy, some experiencing ةيدعلما تانئاكلا ةدحو يف ةيعاجرتسلاا ةساردلا هذه تيرجُأ :ةقيرطلا
،ةدج ،زيزعلا دبع كللما ةعماج يف ةيبطلا ثوحبلل دهف كللما زكربم ةصالخا Results: Based on protease region (PR), genotypes of 63
2004 سطسغأ نم ةرتفلا للاخ كلذو ةيدوعسلا ةيبرعلا ةكلملما samples were as follows: C:22, G:21, B:9, CRF02_AG:5, فيرعتلا ماظن مادختساب ةذوخألما امزلابلا تانيع ليلتح تم دقل .م2009 D:3, A:1, F:1, and J:1. Based on reverse transcriptase ىلإ ةفاضلإاب ،)Celera/Abbott( ةكرش نم )Viroseq 2.5( ينيلجا region (RT), genotypes were as follows: C:23, G:24, .ينيلجا لسلستلا ديدحتل )ABI Prism 3100( ينيلجا لللمحا مادختسا B:9, CRF02_AG: 2, D:2, A:1, and F:1. Antiretroviral تاداضبم جلاعلا اوقلت دق نيذلا ةيدوعسلا ةيسنلجا نم ىضرلما لك ناك دقل susceptibility testing results were as follows: 52% of .جلاعلا مهعم حجني مل مهضعب نأ ريغ ،ةيرقهقلا تاسوريفلا the isolates were susceptible to all 3 major classes of antiretroviral drugs used, 41% had mutations known ةقطنم ىلع ًادامتعا ةينيلجا عاونلأا ميسقت تم ةنيع 63 ينب نم :جئاتنلا
to confer high level resistance to one or more of the C:22، G:21، B:9، CRF02_ :ةيلاتلا عاونلأا ىلإ )PR( زيتوربلا nucleoside analogue reverse transcriptase inhibitors, 16% ًادامتعا ةينيلجا عاونلأا ميسقت تم اميف ،AG:5، D:3، A:1، F:1، J:1 had mutations known to confer high level resistance C:23، G:24، B:9، :يلاتلاك )RT( يسكعلا خسانلا ةقطنم ىلع to non-nucleoside analogues reverse transcriptase تانيعلا ةباجتسا رابتخا رهظأو .CRF02_AG:2 D:2، A:1، F:1 inhibitors, 13% had mutations known to confer high نم )52%( تباجتسا :ةيلاتلا جئاتنلا ةيرقهقلا تاسوريفلا تاداضلم level resistance to one or more of the protease inhibitors (PI). Most isolates were susceptible to 2 or at least one 41%( توتحاو ،تاداضلما نم ةيسيئرلا ةثلاثلا تاعومجملل تلازعلا class of antiretroviral, and only 3% of the isolates had دحاول ةمواقلما نم ةيلاع ةجرد ببست يتلا تارفطلا ضعب ىلع تلازعلا نم resistance to several members of all 3 classes. توتحا اميف ،دياسولكوينلاب ةهيبشلا يسكعلا خسانلا تاطبثم نم رثكأ وأ ةمواقلما نم ةيلاع ةجرد ببست يتلا تارفطلا ىلع تلازعلا نم )16%( Conclusion: Antiretroviral resistance is not uncommon
)13%( توتحاو ،دياسويلكوينلاب ةهيبشلا ريغ يسكعلا خسانلا تاطبثلم in Saudi patients on antiretroviral therapy. وأ دحاول ةمواقلما نم ةيلاع ةجرد ببست يتلا تارفطلا ىلع تلازعلا نم وأ ينتعومجلم تلازعلا رثكأ تباجتسا دقل .زييتوربلا تاطبثم نم رثكأ Saudi Med J 2010; Vol. 31 (9): 987-992
3% نأ ريغ ،ةيرقهقلا تاسوريفلا تاداضم نم لقلأا ىلع ةدحاو ةعومجم .ثلاثلا تاعومجلما ءاضعأ نم ديدعلا ةمواقبم تماق طقف تلازعلا نم From the Special Infectious Agent Unit (Jamjoom, Azhar), King Fahd Medical Research Center and Department of Medical Laboratory Technology, Faculty of Applied Medical Sciences, and Departments of ةعئاش دعت ةيرقهقلا تاسوريفلا تاداضم ةمواقم نأب ةساردلا ريشت :ةتماخ
Internal Medicine (Madani), Hematology (Hindawi, Damanhouri), .تاداضلما هذهب جلاعلا نوقلتي نيذلا ينيدوعسلا ىضرلما ينب Faculty of Medicine, King Abdulaziz University, and Department of Pediatrics (Bakhsh), Maternity & Children Hospital, Ministry of Health, Objectives: To analyze antiretroviral drug resistance and
determine the genotype of human immunodeficiency Received 18th April 2010. Accepted 2nd August 2010. virus (HIV)-1 in Saudi patients by sequencing an Address correspondence and reprint request to: Prof. Ghazi A. Jamjoom, Department of Medical Laboratory Technology, King Fahd Medical Research Center, PO Box 80216, Jeddah 21589, Kingdom Methods: This retrospective study analyzed data from
of Saudi Arabia. Tel. +966 505351850. Fax. +966 (2) 6952059. plasma samples submitted for genotypic drug sensitivity Genotype and drug resistance of HIV-1 . Jamjoom et al Development of resistance to antiretroviral drugs Jeddah, and HIV/acquire immune deficiency syndrome constitutes a threat to patients undergoing (AIDS) outpatient clinic (no samples were received treatment and a continuous concern to their doctors. from expatriate patients). Patients had chronic HIV The transmission of resistant viruses is also a concern infection and were on antiretroviral therapy. Regimens to the community in general.1 Drug resistance assays of antiretroviral drugs used for all categories (adults, have become an important tool for the management pregnant women, children, and neonates) were of human immunodeficiency virus (HIV) infected according to the current updates of the Guidelines of subjects.2-5 Many studies6-11 have been carried on the Panels on Antiretroviral Therapy, Department of antiretroviral resistance in various countries. Reports on Health and Human Services (USA).13-15 The backbone of drug resistance from newly infected patients in Europe therapy was 2 nucleoside analogue reverse transcriptase revealed resistance rates of 9-21%,6-9 while a study from inhibitors (NRTIs) such as zidovudine, stavudine, or the USA10 on newly infected patients indicated a rate didanosine plus lamivudine, combined with a protease of 12.4% phenotypic resistance, and a rate of resistance inhibitor (PI) usually lopinavir/ritonavir, atazanvir, or mutations (genotypic resistance) of 8-22.7%, while nelfinavir, or a non-nucleoside reverse transcriptase multi-drug resistance increased up to 10.2%. Another inhibitor (NNRTIs), such as efavirenz or nevirapine. USA study11 indicated that as many as 76% of viremic Routine investigations for all patients before initiation of patients receiving antiretroviral therapy are infected with antiretroviral therapy (ART) include quantitative HIV viruses that express resistance to at least one of the available polymerase chain reaction (PCR) (viral load). Patients antiretroviral drugs. Resistance to a drug was found to receiving antiretroviral therapy for at least 6 months, be associated with reduced responsiveness to the drug and failing to achieve undetectable viral load were in vivo.4,5 Two types of assays are used for antiretroviral suspected to have antiretroviral resistance. Suboptimal susceptibility testing: phenotypic assays, which measure response to treatment as judged clinically, and/or high the susceptibility of the virus to various drugs in tissue- or increased viral load were the main reasons for ordering culture systems; and genotypic assays, which detect the drug resistance testing. Inclusion criteria for analyzed presence of resistance mutations by sequence analysis samples were Saudi nationals, and viral load >1000 of selected regions of the viral genome.2,12 Both are copies of HIV ribonucleic acid (RNA)/ml. Samples used, each having certain advantages and disadvantages. with low copy number, as judged by electrophoretic Phenotypic assays provide quantitative resistance band intensity upon amplification, and viral load were information and information on new drugs for which excluded from further analysis. Plasma samples from 63 genetic correlates have not been established. However, patients were analyzed at the Special Infectious Agent they are more complex to perform, costly, and time Unit, King Fahd Medical Research Center (KFMRC) consuming, which limits their availability. Genotypic of King Abdulaziz University, Jeddah from August assays are relatively easier to perform, have a quicker 2004 to June 2009. Ethical approval for the study was turnaround time, and are more widely available.2 These obtained from the Medical Ethics Committee at the assays usually involve amplification and sequencing of Faculty of Medicine, King Abdulaziz University. The the reverse transcriptase (RT) and protease (PR) genes, Viroseq2.5® kit (Celera/Abbott, Alameda, CA, USA) and their analysis for known resistance mutations from was used with the ABI Prism 3100 sequencer (Applied widely used data banks. We aimed to shed light on the Biosystems, Carlsbad, CA, USA) for sequencing all extent of HIV drug resistance in Saudi patients. Such isolates. Processing was according to the manufacturers information will be useful for guiding antiretroviral instructions. Viral RNA was purified and reverse- therapy. In addition, defining the most prevalent transcribed into cDNA. A 1.8 kb fragment of the pol genotypes in Saudi Arabia would give us better insight gene of the HIV-1 genome spanning the entire PR on the source and transmission of HIV in this country. region, and approximately two-thirds of the RT region was amplified by the PCR. Within this fragment, a l.3 Methods. All patients were Saudi nationals treated kb region was sequenced. This regions comprises the
as inpatients or outpatients at the King Saud Hospital, entire PR (codons 1-99), and a fragment (codons 1-335) of the RT. An ABI Prism 3100 (Applied Biosystems, Carlsbad, CA, USA) is a capillary-based automated Disclosure. This study was supported by the Chair of
fluorescent sequencer that relies on Sanger’s method Abdullah and Saeed Binzagr for AIDS Research and incorporating dye-labelled ddNTP terminators during Control, and the Ministry of Health, Kingdom of Saudi DNA extension. The obtained sequence was compared on-line for resistance interpretation using the Stanford HIV Drug Resistance Report (http://hivdb.stanford.
Saudi Med J 2010; Vol. 31 (9) www.smj.org.sa Genotype and drug resistance of HIV-1 . Jamjoom et al edu). Susceptibility results were reported for the 3 major such as M184V confer resistance to several drugs classes of antiretroviral drugs, such as NRTI, NNRTI, particularly of the same class (such as, NRTI). For the 4 drugs, efavirenz, atazanavir, ritonavir, and saquinavir, The Excel Program (Microsoft, Redmond, high level resistance was due to the combination of Washington, USA) was used for data handling and mutations each recognized when present alone only to cause intermediate, or low level resistance. Table 4 shows
the frequency of high-level drug resistance mutations Results. The procedure used in this study for both among the 63 samples analyzed. Thirty-three samples
genotyping and antiretroviral resistance determination (52%) had no such mutations detected. Table 5 lists
is based on the amplification and sequencing of the high-level mutations encountered in individual patients whole PR gene, and two-thirds of the RT gene of on different antiretroviral regime. Most mutations HIV-1. These genes code for the 2 main enzymes encountered are well recognized for their occurrence that are targeted by most current antiretroviral drugs. during long-term antiretroviral therapy.
Moreover, they each contain sufficient variability so as to be used for genotyping of various HIV-1 isolates. Discussion. Antiretroviral drug resistance testing
Table 1 shows the genotypes among the 63 Saudi is recommended in acute and early infection before
patients. As can be seen, genotypes C and G were initiation of therapy, in treatment initiation of chronic most common constituting 33-38% each. Genotype HIV infection, in all cases of treatment failure in patients B was less frequent (14%). Circulating recombinant on antiretroviral therapy, and in pregnancy.1,2 Resistance form CRF02_AG was also detected at low frequency. testing may also be considered in other situations, such These results indicate that Saudi patients were infected as for post-exposure prophylaxis where testing is carried by several HIV-1 genotypes. Agreement between the PR-based and RT-based genotypes, which was observed Table 1 - Number of HIV-1 PR-and RT-genotypes among 63 patients.*
in 57 (90.5%) isolates serves as a double-check of the genotyping results for both of these genes. However, Genotype
PR-based*
RT-based*
there was mismatching in 6 (9.5%) isolates. Table 2
genotype
genotype
shows the results of antiretroviral susceptibility testing. Susceptibility to all 3 categories of antiretrovirals was 52% for all isolates. Resistance to NRTI (41%) was most common perhaps reflecting the longer experience with CRF02_AG
the use of these drugs. Resistance to NNRTI (usually considered for the whole class of these drugs) was 16%, and resistance to PI was 13%. Resistance to 2 classes or even the 3 classes of antiretroviral drugs was also observed. Resistance to NRTI plus NNRTI amounted to 8%, while resistance to NRTI plus PI amounted to *Total mismatches between PR and RT genotypes: 6, PR - protease 14%. Thus, dual class resistance amounted to 22%. No gene, RT - reverse transcriptase (polymerase) gene, simultaneous resistance to NNRTI and PI was observed Three percent of the isolates were simultaneously resistant to all 3 categories of antiretrovirals. Table 3 lists Table 2 - Pattern of antiretroviral resistance.
antiretroviral drugs and well-known mutations that were detected in this study, which confer high-level resistance Category
to each drug. Additional mutations, or combinations thereof are recognized to give intermediate, or low level High-level resistance to at least one NRTI resistance to particular drugs. Only mutations causing high level resistance are listed in this table. Those causing intermediate or low level resistance are too numerous to list. Table 3 indicates the frequency (percentage) of
high, intermediate, or low level resistance among our isolates based on mutations that they contain. Common mutations conferring high level resistance include: NRTI - nucleoside analogue reverse transcriptase inhibitors, NNRTI- M184V, T215F/Y, M41L, K65R, T69D, K103N, non-nucleoside analogues reverse transcriptase inhibitors, PI - protease V106A, Y181I, L90M, and I54V. Certain mutations, www.smj.org.sa Saudi Med J 2010; Vol. 31 (9) Genotype and drug resistance of HIV-1 . Jamjoom et al Table 3 - Resistance to individual antiretroviral drugs.
Anti-HIV
Mutations
high-level
intermediate-
low-level
resistance
level resistance
resistance
3TC - Lamivudine, ddC - Zalcitabine, AZT - Zodovudine, ddI - Didanosine, d4T - Stavudine, ABC - Abacavir, TDF - Tenofovir, FTC - Emtricitabine, DLV - Delavirdine, EFV - Efavirenz, ETR - Etravirine, NVP - Nevirapine, ATV/r - Atazanavir+ritonavir, DRV/r - Darunavir+ritonavir, FPV/r - Fosamprenavir+ritonavir, IDV - Indinavir, LPV/r - Lopinavir+ritonavir, RTV - Ritonavir, NFV - Nelfinavir, SQV - Saquinavir, TPV/r - Tipranavir+ritonavir out on the source of infection.1,2 Resistance testing detected, further complicating therapy. As our study was should help the clinician to avoid unnecessary switching conducted on chronically infected patients already on of drugs, rule out adherence problems, perform well- antiretroviral therapy, further studies on treatment naive directed switches rather than empirical changes of drugs, patients are indicated. Some of the frequent mutations use active drugs for longer periods of time, save costs revealed in this study, which are associated with NRTI associated with switching drugs, and avoid unnecessary resistance are: M184V, which confers high-level toxicities from inactive drugs.2 Antiretroviral therapies resistance to lamivudine, it is the main mutation that is with major RT and PI, as well as combination drugs observed in most viruses resistant to treatment with this have been available for a number of years in Saudi drug.1,3 The M41L, T215Y, T215F, K70R confer high Arabia.16 These drugs are given free of charge to Saudi level resistance to zidovudine, and low level resistance nationals. It is expected under these circumstances that to stavudine, didanosine, and abacavir.1,3 The K65R antiretroviral resistance will develop.11 mutation confers resistance to zalcitabine, abacavir, and The current study indicates that HIV resistance to any tenofovir, but not by zidovudine. Several mutations of the 3 major classes of drugs used in Saudi Arabia is observed are associated with NNRTI resistance. The significant, and must be taken into consideration in the K103N mutation is associated with resistance to treatment of chronic HIV infected patients. Furthermore, efavirenz, delavirdine, and occasionally nevirapine; this simultaneous resistance to 2 or all 3 classes of drugs is mutation may confer high level in vitro resistance to Saudi Med J 2010; Vol. 31 (9) www.smj.org.sa Genotype and drug resistance of HIV-1 . Jamjoom et al Table 4 - Frequency of high-level drug-resistance mutations.
Table 5 - Antiretroviral regimens in patients with high level resistance
Mutation
Frequency
Treatment regimen
Mutations associated with resistance to
AZT/ 3TC) + (LPV/r
NRTI
Mutations
AZT / 3TC) + NFV
Mutations
Mutations associated with resistance to
NNRTI
AZT / 3TC) + EFV
Mutations
Mutations associated with resistance to PI
AZT / DDI + NFV
Mutations
AZT / 3TC) + NVP
*total number of patients - 63, NRTI - nucleoside analogue reverse transcriptase inhibitors, NNRTI - non-nucleoside analogues reverse transcriptase inhibitors, PI - protease inhibitors (AZT / 3TC) + IDV
(TDF/FTC) + EFV
these drugs.2 This explains the simultaneous resistance AZT + DDI + EFV
to NNRTIs observed. The Y181I and Y181C mutations are often associated with resistance to nevirapine. The AZT + DDI + IDV
V106A mutations accumulate during ineffective therapy with most NNRTI. The following observed mutations AZT + 3TC + IDV + RTV
have been associated with resistance to PR inhibitors: L90M is observed during failure of therapy with most protease inhibitors; D30N is associated with nelfinavir n - number out of the total 26 patients, AZT - Zidovudine, 3TC - Lamivudine, NFV - Nelfinavir,; EFV - Efavirenz, TDF - Tenofovir, resistance; I54V is frequently found after prolonged NVP - Nevirapine, FTC - Emtricitabine, IDV - Indinavir, RTV - Ritonavir, LPV/r - Lopinavir +Ritonavir. Brackets indicate combined This study indicates that the most frequent HIV-1 genotypes are types C and G (35-38%), while type B only constituted 14%. Non-B subtypes are dominant in Africa and Asia. This favors an African or Asian source circulating in South East Asia.18 The high percentage of for most infections in Saudi patients. Genotype C of genotype G in our findings was unexpected. In addition, HIV-1 is the most frequent genotype in Africa and no CRF-01_AE genotype was detected, while several India, while genotype B is more prevalent in North CRF-02_AG isolates were found. It should be pointed America and Western Europe.12,17,18 However, non-B out that genotyping based on pol gene sequences genotypes have become increasingly more prevalent may differ from that based on env gene sequences as in some European countries making it more difficult determined in the above-sited studies. The large African to exclude these countries as a source of our isolates. and Asian communities in Jeddah, and the possibility The most prevalent HIV-1 genotypes globally in 1999 of transmission to nationals can explain the genotypic were genotypes C (56%), A (23%), B (8%), D (5%) distribution observed in this study. Another factor in and CRF01_AE (5%).17 The CRF-01_AE is widely the transmission of non-genotype B is that, while B www.smj.org.sa Saudi Med J 2010; Vol. 31 (9) Genotype and drug resistance of HIV-1 . Jamjoom et al genotype is dominant among homosexuals,15 HIV is 5. Haubrich R, Demeter L. International perspectives on predominantly transmitted heterosexually in Saudi antiretroviral resistance. Clinical utility of resistance testing: retrospective and prospective data supporting use and current recommendations. J Acquir Immune Defic Syndr 2001; 26:
The database available for comparison of new mutations, and which is used in this study is based 6. Yerly S, Vora S, Rizzardi P, Chave JP, Vernazza PL, Flepp M, on genotype B strains that are prevalent in the United et al. Acute HIV infection: impact on the spread of HIV and States and Europe. This may cause overlooking some transmission of drug resistance. AIDS 2001;15: 2287-2292.
important differences in drug susceptibility with other 7. UK Collaborative Group on Monitoring the Transmission of HIV Drug Resistance. Analysis of prevalence of HIV-1 drug genotypes. It has been reported that some genotypes resistance in primary infections in the United Kingdom. BMJ
of HIV-1 can be less susceptible to PI or NNRTIs than genotype B.19 This includes genotypes C and G, 8. Descamps D, Chaix ML, Andre P, Brodard V, Cottalorda J, which are quite prevalent in our population. Therefore, Deveau C, et al. French national sentinel survey of antiretroviral judgment must be exercised in adopting therapeutic drug resistance in patients with HIV-1 primary infection and in antiretroviral-naive chronically infected patients in 2001-2002. regimens based on genotype B pattern of sensitivity. As J Acquir Immune Defic Syndr 2005; 38: 545-552.
some controversy remains on this issue, it is useful to 9. Yerly S, Von Wyl V, Ledergerber B, Boni J, Schupbach J, collect additional data. This study was limited to Saudi Burgisser P, et al. Transmission of HIV-1 drug resistance in nationals who only represent one fifth to one fourth Switzerland: a 10-year molecular epidemiology survey. AIDS
the total HIV-infected population in Saudi Arabia, the 10. Little SJ, Holte S, Routy JP, Daar ES, Markowitz M, Collier rest being expatriates from various countries mainly AC, et al. Antiretroviral-drug resistance among patients recently in Africa, Asia, and the Middle East.16 The study of infected with HIV. N Engl J Med 2002; 347:385-394.
resistance in the expatriate in comparison to the native 11. Richman DD, Morton S, Wrin T, Hellmann N, Berry S, population may add important information to drug Shapiro MF, Bozzette SA. The prevalence of antiretroviral drug resistance in the United States. AIDS 2004; 18: 1393-1401.
resistance for all groups in this country. In addition, the 12. Hoffmann C, Rockstroh JK, Kamps BS, editors. HIV resistance rapidly changing dynamics of HIV resistance necessitate testing. HIV Medicine. 15th ed. Paris (France); Flying that resistance data be regularly updated. Unfortunately, this is restricted by the relatively high cost and technical 13. Panel on Antiretroviral Guidelines for Adults and Adolescents. requirements of the test. However, this may soon change Guidelines for the use of antiretroviral agents in HIV-1-infected adults and adolescents. Department of Health and Human with newer sequencing techniques that are becoming Services. December 1, 2009; 1-161. Available at http://www.
more widely available, giving substantial savings in cost aidsinfo.nih.gov/ContentFiles/AdultandAdolescentGL.pdf and time.20 Updating of resistance data in treatment- 14. Working Group on Antiretroviral Therapy and Medical experienced patients, and the inclusion of treatment of Management of HIV-infected Children. Guidelines for the use of antiretroviral agents in Pediatric HIV Infection February 23, native Saudi patients and expatriates must be considered 2009; pp1-139. Available from URL: http://www.aidsinfo.nih.
15. Panel on Treatment of HIV-Infected Pregnant Women and Acknowledgment. The authors gratefully acknowledge Mr.
Prevention of Perinatal Transmission. Recommendations Raed Badera, Mr. Badr Masri, and Mr. Azad Qudus, and Ms. Lina for Use of Antiretroviral Drugs in Pregnant HIV-1-Infected Bajri for excellent technical help and for Dr. Sanaa Flimban, Director of Women for Maternal Health and Interventions to Reduce the National AIDS Program in Saudi Arabia for the useful discussions. Perinatal HIV Transmission in the United States. May 24, 2010; pp 1-117. Available from URL: http://www.aidsinfo.nih.
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Saudi Med J 2010; Vol. 31 (9) www.smj.org.sa

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