1

High prevalence of polymorphism in HIV1 variants circulating

in Latvia

High prevalence of polymorphism in HIV1 variants circulating

in Latvia

D.Dusacka, L.Guseva, T.Kolupajeva, J.Vrublevska, I.Januskevica, J.Storozenko, B.Rozentale

Infectology Center of Latvia, Riga, Latvia

D.Dusacka, L.Guseva, T.Kolupajeva, J.Vrublevska, I.Januskevica, J.Storozenko, B.Rozentale

Infectology Center of Latvia, Riga, Latvia

2

Infectology Center of Latvia

1890 Riga City Council decided to build a leper hospital in Riga. The founder initiator –surgeon, Dr.med. Adolf von Bergman.

1891-1937 Riga Leper Hospital for 40-80 patients

1941-1945 Riga Hospital for Special Infectious Diseases with 240 beds.

1945-1982 Riga Hospital for Infectious Diseases

1982 Republic’s Hospital for Clinical Infectious Diseases

1996 State Infectology Center, restructured later as state company “Infectology Center of Latvia”

2004 State agency “Infectology Center of Latvia” under the supervision of the Ministry of Health• 200 beds and more than 35 000 outpatient visits per year• Reference laboratory for microbiology including HIV/AIDS

3

1987 – 2009 Cumulative number of known HIV cases is 4401, 665 patients are on AIDS stage, 428 patients died (217 – on AIDS stage)2939 patients registered at the ICL (67%)

HIV/AIDS in Latvia (by 31.03.2009)

Public Health Agency

4

1990 - AZT monotherapy was started, 1996 – HAART, 336 patients are currently getting treatment2003 - Resistance testing– by hybridization-based kit (Versant HIV1 Inno-LiPa,

InnoGenetics)– since 2006 - by sequencing – based kit (TRUGENE

HIV1 BayerHealthCare-diagnostics )

2006 - Resistance testing before starting therapy and in case of treatment failures

(HIV Treatment Guidelines, ICL, Riga, 2006)

HIV/AIDS in Latvia (by 01.03.2009)

5

To detect subtypes circulating in Latvia in 2006-2008to detect prevalence of HIV drug resistance associated mutations (RAMs) – in treatment – naïve– treatment- experienced patients

to analyze the susceptibility of HIV circulating in treatment – naïve and treatment-experienced patients to different drugs groups

Aim of study

6

Methods: population

Data of HIV genotyping, performed in 2006-2008 for clinical reason:– 126 treatment-experienced individuals with

treatment failures– 99 treatment-naïve individuals were tested before

starting therapy Data of HIV genotyping, performed in 2007-2008 in the frame of the EHR (European HIV Resistance ) project :– 34 treatment-naïve individuals

7

Characteristics of the study population

Characteristics Treatment naïve

Treatment experienced Total

Number 133 126 259Age 2 - 66 y. 1 - 79 y. 1 - 79 y.Sex 92♂ / 41♀ 80♂ / 46♀ 172♂ / 87♀Heterosexual 46 30 76MSM 11 28 39IDU 75 59 134Vertical transm. 1 7 8Unknown 0 2 2

8

Methods: TRUGENE HIV-1

Sequencing of pol genes:PR codons 4-99RT codons 38-247Interpretation according to TRUGEN expert

BayerHealthCare-diagnostics

9

Subtyping was performed by using Rega 2.0. data base

Phylogenetic analyses were conducted using MEGA version 4 (Tamura, Dudley, Nei, and Kumar 2007)

References sequences from the HIV sequence database at Los Alamos

RAMs (Resistance Associated Mutations) inclusion criteria for estimation of prevalence:

– IAS (International AIDS Society) mutation list (2008),that is used often in surveillance studies in Europe

– according to R.W. Shafer et al. (2007)

Methods

10

Phylogenetic analyses by MEGA v.4

References sequences from the HIV sequence database at Los Alamos:

A1.RU.2003.03RU200613A1.RU.2000.RU00051A1.BY.1997.97BL006AF193275A1.UA.2000.RU98UA0116

B.RU.2004.04RU139089B.RU.2004.04RU139095B.RU.200404RU129005B.RU.2004.04RU128005

03AB.RU.97.KAL1532.AF19327603.AB.RU.1998.RU9800103.AB.BY.2000.98BY1044306_cpxRU.2005.04RU001

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11

Results of HIV subtyping

83%

15% 2%

Treatment naïve group Treatment experienced group

Most common HIV-1 subtypes in our study were subtypes A -77.2% and B – 20.8%Rare subtypes:- treatment naïve: CRF02AG, CRF03AB, CRF06CPX- treatment experienced: C

72%

26%2%

Subtype A Subtype B Others

12

Transmission ways

Treatment naïve(n=133)

Treatment experienced (n=126)

HIV-1 subtypes HIV-1 subtypesA1 B others A1 B others

IDU 71 3 - 59 0 0MSM 1 7 - 2 25 -Hetero 36 7 3 22 6 2Bisexual - 3 - - 1 -Vertical 1 - - 7 - -Unknown 1 - - - 2 -Total 110/83% 20/15% 3/2% 90/71% 34/27% 2/2%

Results of HIV subtyping

13

94.7%

5.3% 30%

70%

RAMs Prevalence in Treatment naïve GroupAccording to IAS

mutation list (2008)According to R.W. Shafer

et al. (2007)

Class RAMs n Predicted drug resistance

NRTI M184V 2 Lamivudine (3TC)

A62V 40 Cause only in combination with Q151M

NNRTI K103N 2

Nevirapine (NVP), Efavirence (EFV)K103N+G190S 1

K103N+P225H 1

NRTI+NNRTI M184N+K101E+G190S 1 Lamivudine (3TC) and Nevirapine (NVP), Efavirence (EFV)

MutantWild

14

Prevalence of polymorphic mutations among treatment naïve patients (n=133)

RAMsA subtype

(n=110/83%)B subtype

(n=20/15%)Others

(n=3/2%)n % n % n %

Only A62V 5 4,5 1 5 0 0

Only V77I 3 3 8 40 1 2

A62V + V77I 34 31 0 0 0 0

15

RAMsA subtype

(n=91/72%)B subtype

(n=33/26%)Others

(n=2/2%)

n % n % n %

Only A62V 1 1 1 3 0 0

Only V77I 5 6 10 29 0 0

A62V + V77I 23 26 0 0 0 0

Prevalence of polymorphic mutations among treatment experienced patients (n=126)

16

LV.0

825.

IDU.

t.A62

V.V7

7ILV

.255

8.ID

U.tn

.A62

V.V7

7IA1

.RU.

2003

.03R

U20

06 1

3LV

.151

4.H

E.t.A

62V.

V77I

LV.1

041.

IDU.

tn.A

62V.

V77I

LV.1

135.

IDU.

tn.A

62V.

V77I

LV.1

532.

IDU.

tn.V

77I

LV.2

068.

HE.t.

V77I

LV.1

224.

IDU.

tn.A

62V.

V77I

LV.1

614.

IDU

.t.A

62V.

V77I

LV.1

625.

HE.tn

.A62

V.V7

7I

LV.22

84.H

E.t.A62

V.V77

I

LV.145

4.HE.tn

.A62

V.V77I

LV.146

5.HE.tn

.A62V

.V77

I

LV.2492 .IDU .t .A

62V.V77 I

LV .2554.IDU.tn

.A62V.V

77I

LV .2 096.HE .t.A

62V.V77I

LV.251 7.IDU.t.A

64V .V7 7I

LV .0635 .IDU .t .A

62V .V 77 I

LV .11 22. IDU.t.A

62 V.V 77I

L V.154 2. IDU .tn .A 62V .V7 7 I

L V .1028 .ID U .t .V 77 I

L V .10 35 . ID U . tn .A 62 V.V 77 I

L V .2 3 1 7 .H E . tn .A 6 2 .V 7 7 I

L V .2 3 6 1 .H E . tn .A 6 2 V .V 7 7 I

L V .2 6 8 2 . H E .t n. A 6 2 V

L V .2 8 3 0 .H E . tn .A 6 2 V .V 7 7 I

L V .1 1 3 9 .H E .t .A 6 2 V .V 7 7 IL V .2 6 9 3 .H E .t n .A 6 2 V .V 7 7 IL V .1 6 3 8 . ID U . tn .A 6 2 V . V 7 7 IL V .2 6 1 1 .H E . tn .A 6 2 V .V 7 7 I

L V .2 3 9 6 .N E Z . t.A 6 2 V . V 7 7 I

L V .0 7 2 8 . ID U . t.A 6 2 V .V 7 7 I

L V .1 7 7 6 .ID U .t n .A 6 2 V .V 7 7 I

L V .0 7 71 . ID U . t.A 6 2 V .V 7 7 I

L V .2 537 .H E . tn .A 62V .V7 7 I

L V.066 7. ID U .t.A 62V .V7 7I

LV.267 8.HE .tn.A6 2V .V 77I

LV .1059 .IDU .tn .A 62V .V 77 I

LV.268 5.HE .tn.A6 2V .V77I

LV .0716 .IDU .t .A 62V .V77 I

LV.2127.IDU.t.A 62V .V77I

LV.2677 .IDU .tn.A62V.V77 I

LV.1045.IDU.tn.A62V

A1.RU.2000 .RU00051

A1.UA.2000.98UA0116

A1.BY.1997.97BL006 AF193275

LV.0389.IDU.tn.A62V

LV.2227.HE.t.A62V

LV.0128.IDU.t.V77I

LV.2453.HE.tn.V77I

LV.1

080.

IDU

.tn.A

62V.

V77I

LV.2

659.

IDU.

tn.A

62V.

V77I

LV.1

022.

IDU.

tn.A

62V.

V77I

LV.2

271.

IDU.

tn.A

62V.

V77I

LV.1

160.

IDU

.t.A

62V.

V77I

LV.2

056.

IDU.

t.A62

V.V7

7ILV

.071

2.ID

U.tn

.A62

V

LV.1

042.

HE.tn

.A62

V

LV.1

446.

IDU

.tn.V

77I

LV.1

530.

IDU.

t.V77

I

LV.0

937.

IDU.

t.A62

V.V7

7I

LV.2

308.

IDU.

t.A62

V.V7

7I

LV.0

930.

IDU.

tn.A

62V.

V77I

LV.2

656.

HE.tn

.A62

V

LV.1

129.

IDU.

t.V77

I

LV.25

82.ID

U.tn

.A62

V.V7

7I

LV.2

515.

IDU.tn

.A64

V.V77

I

LV.06

68.ID

U.tn.A

62V.

V77I

LV.A003

.06.

VERT.A62V

.V77

I

LV.0191.HE.tn

.V77I L

V .1468.IDU.tn

.A62V.V

77I

A 1.UG.98.98UG57136.A

F48 4509

A2 .CD.97.97C DKTB 48.A

F28 6238

A 2.C Y.94 .94 CY 017 41 .A F2862 37

A1 .K E.94.Q 23 17 .A F0048 85

A1 .S E .94 .S E 7253 .A F 0696 70

A 1.U G .92 .92U G 037 .U 5 1190

03 A B .R U .199 7 .K A L 15 3 2

0 3 A B .R U .9 7 .K A L 1 5 3 2 .A F 1 9 3 2 7 6

0 3 A B .B Y .2 0 0 0 .9 8 B Y 1 0 4 4 3

0 3 A B .R U .1 9 9 8 .R U 9 8 0 0 1L V .2 6 3 3 . ID U . tn .A 6 2 VL V . 2 4 3 7 . ID U . tn .V 7 7 IB .R U . 2 0 0 4 .0 4 R U 1 2 8 0 0 5

L V .0 2 4 1 . H O . t. A 6 2 V

L V . 2 0 3 1 .H O .t n .V 7 7 I

L V . 2 3 3 0 .B I. tn . V 7 7 I

B .R U .2 0 0 4 .0 4 R U 1 3 9 0 8 9

B .R U .2 0 0 4 .0 4 R U 1 3 9 0 9 5

L V .2 6 72 . ID U . tn .V 7 7 I

B .R U .20 04 .04R U 129 005

L V .04 86 .B I .t .V 77 I

LV .00 12.HO .t.V 77I

LV .0014 .H O .t .V 77 I

LV.004 2.HO .t.V7 7I

LV.005 0.HO.t.V7 7I

LV.143 2.HE.t .V77I

LV.0113.HO.tn.V77 I

LV.2705.HE.tn.V77 I

LV.2288.HO.t.V77I

LV.2563.BI.tn.V77I

LV.2010.HO.tn.V77I

LV.2269.BI.tn.V77I

06 cpx.RU.2005.04RU

001

N.CM.02.DJO

0131.AY532635

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.98.98CM

U2901.AY169812

0 . 0 5

Phylogenetic analyses of sequences with A62V and/ or V77I

17

Prevalence of polymorphic mutations among treatment naïve patients

15,6

57,0

11,718,8

12,5

87,5

7,8 6,317,2

86,7

35,9

86,7

43,8

0102030405060708090

%

L10I

I13V

I15V

G16

E

K20

R

M36

I

D60

E

I62V

L63P

/T

H69

K

V77I

L89M I93L

18

Most frequently occured RAMs in thetreatment experienced group

19

23

64

13

5 4 5 64

0

5

10

15

20

25M

184V

A62

V

T21

5Y

K70

R

K10

3N

G19

0S

P225

H

L90

M

M46

I

V82

A

NRTI mutation NNRTI mutation PI mutation

%

19

Susceptibility to drugs amongtreatment experienced patients

6%

2%

14%

59%

6% 1% 3%9%

Susceptible NRTI NNRTIPI NRTI+NNRTI NRTI+PINNRTI+PI NRTI+NNRTI+PI

20

Conclusions:

1. Most common HIV-1 subtypes in our study were subtypes A -77.2% and B – 20.8%, that agree with previously published data for Latvia.

2. Prevalence of drug resistant virus in treatment-naive HIV1 infected persons was estimated as 5.3 %. These data are comparable with available data for the Europe.

3. Drug resistance was predicted in 31% of treatment experienced individuals.

21

4. High frequency of polymorphism in HIV1 subtype A at protease and RT sites was detected in treatment-naïve group: A62V - 35.5%, that occurs mostly in combination with V77I (31%). Sequences with A62V and V77I combination were closely related.

5. In HIV1 subtype B A62V was detected rare (5%), combination A62V and V77I was not observed.

6. Others most frequent polymorphisms in subtypes A and B were M36I – 87,5 %, H69K -86,7 %, L89M -86,7 %, I13V- 57 %.

Conclusions:

22

Thanks for your attention !