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SURVEILLANCE OF TRANSMITTED HIV-1 DRUG RESISTANCE IN GONDAR TOWN USING WHO PROTOCOL
RETROVIRUSES AND NOVEL DRUGSJUNE 08-09, 2015
CHICAGO
DAWIT ASSEFA
ETHIOPIAN PUBLIC HEALTH INSTITUTE LUND UNIVERSITY
Source: EPP/Spectrum HIV estimates, EPHI, 2014
Background…
HAART is the recommended form of treatment
HAART is usually combination of at least 3 drugs
2 NRTIs + 1 NNRTI or 1-2 PIs
Standard ART regimens
Restricted drug options
Limited lab monitoring
Decentralized service
delivery and task shifting
People receiving ART
12.9 million globally
11.7 million in LMIC
38% of people living with HIV
SuccessART coverage reached 75.4% in 2013
ART made available in both HCs & HPs since August 2006
# ever started ART increased from 900 at the beginning of 2005 to
743,299 by June 2013.
ART sites rose from 3 in 2005 to 913 by 2013.
2007/8 2008/9 2009/10 2010/11 2011/12 2012/13
Ever enrolled 266507 374835 473772 580919 666147 743299
Ever started 150136 210637 268934 333434 379190 438804
Alive and on ART 110611 153741 207733 247805 274708 309656
50000
150000
250000
350000
450000
550000
650000
750000
Trends on number people receiving HIV/AIDS careN
um
ber
of
peo
ple
Rapid scale up is prone to emergency and transmission of HIVDR
It is very important to remain watchful!
Tracking of transmitted HIV DR is an expensive business
High laboratory costs
WHO recommends minimum-resource method to evaluate
transmitted drug-resistant HIV: Threshold Survey (HIVDR-TS)
Small sample size < 50
And yet reliable results
Can be done periodically
HIVDR Threshold Surveys
• The HIV Drug Resistance Threshold Survey (HIVDR-TS) :
Method supports classification of the prevalence of transmitted
HIV drug resistance in a specific geographic area into 1 of 3
categories:
• LOW prevalence : < 5%
• MODERATE prevalence : Between 5% and 15%
• HIGH prevalence : > 15%
– Method doesn’t estimate point prevalence
– Result not national or clinic specific but apply to the geographical
area surveyed within the country
Pooled analysis from 82
surveys covering a total
of 3588 recently-infected
patient
- 72 surveys with results that could be classified as low, moderate or high
- 20 (28%) reported moderate levels of resistance to any drug class
- Moderate levels of resistance :- 18% in 2004-2006 period to 32% in 2007-2010
Transmitted HIVDR (WHO surveys 2004-2010)
Prevalence of HIVDR in ARV-naïve individuals, since ARV rollout
Gupta et al. Lancet 2012
To undertake HIV drug resistance threshold survey in HIV
Counseling and Testing clients at Gondar University Hospital
and Gondar health center
Objective
Study design
Cross-sectional study
Study site Selection
Based on WHO recommended criterion
Gondar University hospital and Gondar health center
Greater risk factors for emergence of HIVDR
Holly water
Higher number of ART clients
Longer period of ART use
Previous HIVDR report etc..
Methodology
Study participants
• VCT clients at GUH and Gondar HCs
Inclusion criteria
Age between 18-25 and no previous pregnancy if female
Has lived in the Gondar town for about a minimum of a year
Documented positive HIV test result
No previous positive HIV test
No known exposure to antiretroviral drugs
No known AIDS-defining illness
Not eligible to start ART
First risk-defining event within the past 3 years (e.g.,drug injection, STI
Methodology …
DATA COLLECTCTION, MANAGEMENT &
PROCESSING
Methodology …
VCT center• Check eligibility • Consent participants• Draw blood (10ml)• Send participant data to
database• Test specimen for HIV• Send sample to lab
Laboratory• Run CD4 count• Prepare DBS• Separate plasma• Fill Sample transmittal form• Store samples• Send ST form to database
Database• Check Survey codes for identity• Enter data into database• Lock hard copies in a cabinet• Ship data to EHNRI along with samples
EPHI• Receive specimens based the specimen receipt SOP• Synchronize data with existing database• Store specimens at -80
DataData
samples
DATA FLOW
HIVDR report
Sequence data editing
Automated Sequencing
Purification
Cycle sequencing
Purification
Gel electrophoresis
Amplification
RNA extraction QIAamp Viral RNA mini kit
Six primer (3 forward, 3, reverse)
QIAquick purification kit
1% agarose gel electrophorosis
RT-PCR & Nested PCR
Stanford HIVDR database
Recall
ABI 3100/3500xl Genetic analyzer
Isopropanol
IN HOUSE ASSAY
06-99 PR 1-251RTRT-PCRPrtM-F1RT-R1
Nested PCRPrt-F2RT-R2
3 reverse primers
AV44,90V1, RT-R23 forwarded primer
A35V, AV36V, Prt-F2
Forwarded primer Position HXB21. Prm-F1 2057-20852 Prt-F2 2243-22663. A35V 2556-25774. AV36V 2869-2889
Reverse primer Position HXB2 1. RT-R1 3370-33482. RT-R2 3326-33043. AV44 2952-29314. 90V1 2639-2619
CYCLE SEQUENCING
RT-PCR & Nested PCR
06-99 PR 1-251RT
Methodology …
Transmitted HIV-1 Drug Resistance Analysis
• TDR mutations
– Calibrated population resistance (CPR) tool version 6.0 (
http://cpr.stanford.edu/cpr/servlet/CPR )
• Rates and frequencies of ARV drug resistance associated mutations
– Stanford Genotypic Resistance Interpretation Algorithm
http://hivdb.stanford.edu/pages/algs/HIVdb.html
Phylogenetic analysis was done using MEGA 5 & BioEdit (Version 7.0.9.0)
Findings
Study Population
Follows the sequential sampling method selected by WHO for the
surveillance of transmitted HIVDR in low-resource settings
A total of 84 samples were collected
64 from Gondar university hospital
20 from Gondar health center
The mean age of participants was 21 years (range: 18–24 years)
70 (83.3%) were females
Sample Number genotyped (SNG)
Lower Limit (LL)
Running total of specimens with HIVDR (RT)
Upper Limit (UL)
Sample Number genotyped (SNG)
Lower Limit (LL)
Running Total of specimens with HIVDR (RT)
Upper Limit (UL)
1 ND 0 ND 25 ND 0 6
2 ND 0 ND 26 ND 0 6
3 ND 0 ND 27 ND K103N 6
4 ND 0 ND 28 ND G190S 6
5 ND 0 ND 29 ND 2 6
6 ND 0 ND 30 ND 2 6
7 ND 0 ND 31 ND K103N 6
8 ND 0 ND 32 ND 3 6
9 ND 0 ND 33 ND 3 6
10 ND 0 ND 34 1 3 6
11 ND 0 ND 35 1 3 7
12 ND 0 ND 36 1 3 7
13 ND 0 ND 37 1 3 7
14 ND 0 5 38 1 3 7
15 ND 0 5 39 1 3 7
16 ND 0 5 40 1 3 7
17 ND 0 5 41 1 3 7
18 ND 0 5 42 1 3 7
19 ND 0 5 43 1 3 7
20 ND 0 5 44 2 3 7
21 ND 0 5 45 2 3 7
22 ND 0 5 46 2 3 8
23 ND 0 5 47 2 3 8
24 ND 0 5 STOP STOP STOP STOP
Findings …WHO HIVDR threshold category
• 67 (78%) were successfully amplified and sequenced
• HIVDR prevalence was categorized using the WHO TS binomial
sequential sampling method.
• 3 specimens were carrying NNRTI resistance mutations (K103N
G190S, K103N) after genotyping of 47 sequences
• According to the WHO recommendations of sampling and
classification plan prevalence of transmitted HIVDR is classified as
– Moderate prevalence (5% - 15%)
Findings …Prevalence of transmitted HIVDR for each class of ARV
NNRTI moderate (5%–15%)
NRTI low (<5%)
PI low (<5%)
• By analysis of the 67 samples amplified and sequenced
– 4(6%) samples with major HIVDR mutation associated
with NNRTI
– No NRTI and PI associated major mutation
HIVDR among treatment naïve patient in Gondar town
Year of sample collection
Overall NNRTI NRTI PI
Kassu et.al 2003 3/92= 3.3% G190A (n=2) V75I ( n=1) No
Andargachew 2008/09 8/155=5.2% Y188HY (n=1) K101E (n=1) G190A (n=2)
K219E (n=1)L210W (n=2) K65R (n=1)
No
Dawit et al 2011/12 4/67= 6% K103N (n=2)G190S (n=1)Y181Y (n=1)
None No
Discussion• Our results indicated that the levels of TDR to be increasing,
Moderate prevalence (5%–15% ).
Overall 6.0% prevalence of primary HIVDR
• An increasing ARV drug exposure in populations, following the
roll-out of ARVs for treatment and prevention of mother-to child
transmission, may cause a rise in TDR in the study area.
The Ethiopian Health and Nutrition Research
Institute
Discussion …Country Year Prevalence Year Prevalence
Zambia 2005 O% 2009 15.3%
Uganda 2006/7 0% 2009/10 8.6%
Cameron 2001 1.9% 2007 12.3%
Kenya 2005 7.5% 2009/10 13.2%
Discussion …Similar reports
In the Africa region, a more detailed analysis by drug class
showed a statistically significant increase in the prevalence of
NNRTI mutations.
East Africa 36% per year
Southern Africa 23% per year
Estimated prevalence of NNRTI resistance was 5.1% 8 years after
rollout in east Africa.
WHO HIVDR report 2102
• The current results and others clearly showed an increase in
prevalence of DRMs among untreated HIV-1-infected individuals
in Gondar town
• Needs to be confirmed through repetition of the survey in this
area and possibly in additional areas of the country.
• Furthermore work should be done to identify possible sources of
HIVDR transmission,
Conclusion
THANK YOU