Antiretroviral Resistance in HIV-1 Patients at a Tertiary Medical Institute in Saudi Arabia: a Retrospective Study and Analysis. Journal Club, Virology Rotation , 1/5/2019

1. Antiretroviral Resistance in HIV-1 Patients at
a Tertiary Medical Institute in Saudi Arabia:
a Retrospective Study and Analysis
Clinical Microbiology Resident.
King Fahd Hospital of the University.
Teaching Assistant, Department of Microbiology, Imam Abdulrahman
Bin Faisal University, Dammam, Saudi Arabia.
Abdullatif Sami Al Rashed

3. Objectives
Introduction
Material and Methods
Results
Conclusion
Critical Appraisal

5. • Human Immunodeficiency Virus is a retrovirus from the lentivirus
genus.
• It is (+) ssRNA that use Reverse Transcriptase to synthesize DNA
Introduction

7. HIV Replication (HIV Life Cycle)
• HIV binds to CD4 receptors found on macrophages and helper T
lymphocytes in addition to a coreceptor present on these cells.
• The life cycle of HIV is carried out in 6 steps:
(1)Binding and Fusion, (2) Reverse Transcription, (3)
Integration, (4) Transcription, (5) Assembly and (6) Budding.
• The replication cycle of HIV-1 is accomplished in vivo in
approximately 24 hours. (manual of clinical microbiology, 12th edition, 2019)

8. Binding &
Fusion
Budding
Assembly
Integration
Transcription
Reverse
Transcription
Coreceptor

9. https://aidsinfo.nih.gov/understanding-hiv-aids/glossary/1596/life-cycle

10. Provirus

11. Antiretroviral Therapy (ART)
• There are More than 30 antiretroviral (ARV) drugs in seven
mechanistic classes are Food and Drug Administration (FDA)-
approved for treatment of HIV infection.
https://aidsinfo.nih.gov/understanding-hiv-
aids/infographics/25/fda-approval-of-hiv-medicines

12. Antiretroviral Drugs (ARV)
ARV
Nucleosid
e reverse
transcript
ase
inhibitors
Non
Nucleosid
e reverse
transcript
ase
inhibitors
Protease
inhibitors
Integrase
strand
transfer
inhibitors
Fusion
inhibitor
CCR5
antagonis
t
CD4 post-
attachme
nt
inhibitor
+ two drugs, Ritonavir and
Cobicistat that are used as
pharmacokinetic (PK)
boosters to improve the PK
profiles of some ARV drugs

14. Guidelines for the Use of Antiretroviral Agents
https://aidsinfo.nih.gov/

15. Guidelines for the Use of Antiretroviral Agents
https://aidsinfo.nih.gov/ https://aidsinfo.nih.gov/guidelines

16. An antiretroviral (ARV) regimen for a treatment-naive patient generally
consists of two nucleoside reverse transcriptase inhibitors (NRTIs)
administered in combination with a third active ARV drug from one of three
drug classes:
1. An integrase strand transfer inhibitor (INSTI),
2. A non-nucleoside reverse transcriptase inhibitor (NNRTI), or
3. A protease inhibitor (PI) with a pharmacokinetic (PK) booster
https://aidsinfo.nih.gov/

17. HIV Resistance
• HIV-1 genetic variability results from:
• High rate of HIV-1 reverse transcriptase (RT) processing errors
• Recombination when more than one viral variants infect the same cell
• Accumulation of proviral variants during the course of infection.
• Earlier studies measures the HIV mutation rate in vivo have
determined a forward mutation rate of 3 × 10−5 mutations per target
base pair per replication cycle (Mansky L et al, 1995)
• Estimates of HIV mutation rate depend upon a number of factors
including size of viral population and viral fitness of mutant strains.
(Abram et al., 2010; Coffin, 1995; Levy et al., 2004; Mansky, 1996)

18. HIV Resistance
• Studies of HIV from clinical patient isolates suggest that HIV has:
1. Rapid viral turnover (108 to 109 virions per day), (Ho D et al., 1995; Wei x et al., 1995; Pereleson et al., 1996)
2. Large numbers of infected cells (107–108), (Chun T et al., 1997)
3. And a high level of recombination. (Jung A et al., 2002)
• The high mutation rate by HIV generates a genetically diverse set of
viruses usually from a single infecting viral genome.
• The main source for the high mutation rate of HIV-1 is due to the
absence of 3'→5' exonucleolytic proofreading activity of HIV-1 RT (Preston B et
al., 1996; Menendez-Arias L, 2002)

19. ARV Resistance
• ARV Drug Resistance Mutations (DRMs) can be transmitted or
acquired.
• For some ARVs, multiple Drug Resistance Mutations are required to
reduce susceptibility, while for others a single DRM is sufficient. (Abram et al.,
2010; Coffin, 1995; Levy et al., 2004; Mansky, 1996)
• These mutations cause treatment failure and other treatment lines
must be initiated according to the resistance profile.

20. ARV Resistance
• Two types of methods are
available to assay for HIV
resistance:
•Genotyping Assays.
•Phenotyping Assays.

21. Genotyping Assays
• Standard genotypic resistance testing (SGRT) involves the use of
dideoxyterminator Sanger sequencing of protease, RT, and/or
integrase to identify established clinically significant DRMs.
• SGRT is performed on PCR products directly amplified from cDNA that
has been reverse-transcribed from plasma RNA.
• Furthermore, phylogenetic analysis and distance measurements of
sequences can be used to detect possible laboratory contamination
or specimen mislabeling at the point of collection.

22. Genotyping Assays
• Although the lower limit of SGRT detection is generally agreed to be
around 20%, next generation deep sequencing (NGS) technologies
often detect drug-resistant variants at levels well below a 20%
threshold.
• However, the sensitivity and specificty with these ultra-sensitive
assays varies widely between studies. (Jabara et al., 2011; Keys et al., 2015; Natalia
Stella-Ascariz et al, 2018).

23. Genotyping Assays
• Interpreting test results requires knowledge of the mutations selected
by different antiretroviral (ARV) drugs and of the potential for cross
resistance to other drugs conferred by certain mutations.
• The International AIDS Society-USA (IAS-USA) maintains an
updated list of significant resistance-associated mutations in the RT,
PR, IN, and envelope genes. The Stanford University HIV Drug
Resistance Database (https://hivdb.stanford.edu/) also provides
helpful guidance for interpreting genotypic resistance test results.

25. https://www.monogrambio.com/hiv-tests/genotypic-assays
HIV Genotypic Testing

26. Phenotyping Assays
• Phenotypic assays: measure the ability of a virus to grow in different
concentrations of ARV drugs in cell culture.
• Replication of these viruses at different drug concentrations is
monitored by expression of a reporter gene and is compared with
replication of a reference HIV strain.
• The drug concentration that inhibits viral replication by 50% (i.e., the
median inhibitory concentration [IC50]) is calculated, and the ratio of
the IC50 of test and reference viruses is reported as the fold increase
in IC50 (i.e., fold resistance).
Dana S Clutter et al., 2016; manual of clinical microbiology 12th edition

27. https://www.monogrambio.com/hiv-tests/phenotypic-assays
HIV Phenotypic Testing

28. • Because of no genetic profiling of the resistance-causing mutations in the HIV-1
virus have been done nor analyzed previously at Saudi Arabia and especially
KFSH&RC.
• This paper aims to presents an initial report and a profiling survey of HIV-1 DRMs
of patients seen at KFSH&RC.
• Identifying these resistance causing mutations will guide to establish an in house
diagnostic assay for screening of these possible DRM as a diagnostic tool for the
HIV clinic for better patient care and treatment.
AIM:

29. Actually we started with the
lancet journal then went down to
JID then to BMC ID

31. Samples and Data Collection
A retrospective study on Patients treated at KFSH&RC
since 2003 up to 2016.
The drug resistance mutations profiles of 103 HIV
patients at KFSH&RC, who were undergoing highly
active antiretroviral therapy (HAART) protocols were
included in the study.

32. Selection Criteria
Our selection criteria were all HIV-infected adults on
HAART regimens and are experiencing virological
failure (> 500 copies/mL), with resistance mutations
detected by consensus sequencing.
Genotyping testing was done at their reference
laboratory in Mayo clinic.

33. Genotyping
Testing was performed by RT-PCR and DNA sequencing
method (Trugene HIV-1 Genotyping Kit; Siemens Healthcare
Diagnostics, Inc.). Results were based on manufacturer’s
most recent FDA-approved interpretive guidelines.
HIV-1 genotype was analyzed by TRUGENE HIV-1
Genotyping Kit (DNA sequencing assay; Bayer HealthCare
LLC), and interpretive results were based on manufacturer’s
Guidelines v8.0.

34. Analysis
The mutations were reported and their profile for each ART
group of drugs was analyzed.
The comparison was done for single drugs in addition to
known combination therapy. Additionally, the percentage
of primary drug resistance causing mutations reported
annually was also analyzed.

36. PopulationCharacteristic
This study includes 103 HIV-1-infected Saudi patients
enrolled between the years 1988–2016.

37. Prevalence of Drug Resistance
• Mutations were analyzed in relation to demographic, clinical, and
laboratory variables at time of genotyping. The drug resistance is
evaluated through genotyping tests.
• The highest frequency for drug resistance in the study was reported
to the NRTI’s (68%) followed by (47%) for both NNRTI’s and PRI’s, as
seen in Fig. 1.

39. • K103 N was the
main and most
common mutation
detected.
• Interestingly,
mutation I88H,
K101Q, E135G,
V179E and F227 L
are novel mutations
never reported
previously.
Prevalence of Drug
resistance
according to the
class of ART
(NNRTIs)

40. • A range of mutations in
RT including M41 L,
K65R/E/N, D67N, K70R,
I74V, I84V, M184 L, L210
W, T215Y/F, and K219E.
• novel mutations were
also observed, including,
T215C/ D/S/V.
Prevalence of Drug
resistance
according to the
class of ART
(NRTIs)

42. • The use of PI’s
alone also yielded a
number of
mutations, similar
to what is reported
internationally.
Prevalence of Drug
resistance according
to the class of ART
(PI)

43. Prevalence of Drug
resistance according
to the class of ART
(combination of PIs)
• Mutations at codon
82, V82A/ T/F/S, was
reported in our
patients receiving
treatment with
indinavir and ritonavir.

45. Conclusion
• Drug-resistant HIV are increasing in the recent years in the HIV-
infected population and this is caused by the rapid replication rate
of the virus and its characteristic genetic adaptation capabilities.
• Periodical reporting of HIV mutations in the Kingdom of Saudi
Arabia (KSA), will contribute to better therapy and outcome for
the patients. The presence of novel mutations, which were not
reported previously in the international annual report highlights the
importance of these periodic reports.

46. Conclusion
• The prevalence of primary or transmitted HIV drug resistance to
all of the drugs and drug classes that were evaluated in this study
was 66% and 34% respectively.
• These findings provide a useful background for ART in KSA and
contribute reference data for the surveillance of HIV drug
resistance around the world.

47. Critical Appraisal

49. Question Yes No Can’t Tell
Was there a clear statement of the aims of the research?
Is a qualitative methodology appropriate?
Was the research design appropriate to address the aims of the
research?
Was the recruitment strategy appropriate to
the aims of the research?
Was the data collected in a way that addressed the
research issue?
Has the relationship between researcher and
participants been adequately considered?
Have ethical issues been taken into consideration?
Was the data analysis sufficiently rigorous?
Is there a clear statement of findings?
Do the results of this study fit with other available evidence?

50. Critical Appraisal Summary
Positive Points Negative Points
Important topic Methodology was not detailed
Type of Study Typing mistakes
Ethical Approval Data analysis and results are not
sufficient
Novel Mutations were
discovered
They didn’t mention the type of
statistical analysis used and the by
which program
They used FDA
approved kits

51. THANK YOU