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