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STATENS SERUM INSTITUT
Antiretroviral therapy and Antiretroviral therapy and development of resistant HIVdevelopment of resistant HIV
Louise Bruun JørgensenLouise Bruun JørgensenDepartment of VirologyDepartment of VirologyStatens Serum InstitutStatens Serum Institut
STATENS SERUM INSTITUT
STATENS SERUM INSTITUT
Diagnostic Monitorering
* HIV antibodies * HIV RNA (Viral load) (ELISA – WB) (PCR)
* HIV antigen * CD4 counts (ELISA) (FACS)
* HIV DNA * Resistance (PCR) (PCR-sequencing)
Culture
Virological diagnosis and monitoring of HIV-1 infektion
STATENS SERUM INSTITUT
MACS study: 209 pt inrolled in 1984 and 1985
STATENS SERUM INSTITUT
MACS study: 209 pt inrolled in 1984 and 1985
STATENS SERUM INSTITUT
STATENS SERUM INSTITUT
STATENS SERUM INSTITUT
Drugs approved for treatment of HIV
Pommier, Nature Reviews, 2005
STATENS SERUM INSTITUT
NRTI
STATENS SERUM INSTITUT
NNRTI
STATENS SERUM INSTITUT
Protease inhibitors
STATENS SERUM INSTITUT
Protease inhibitors
STATENS SERUM INSTITUT
Treatment of HIV patients
STATENS SERUM INSTITUT
Recommendations for HIV treatment in Denmark
HAART3-4 antiviral drugs
1. EFV + AZT + 3TC2. NEV + AZT + 3TC3. LOP/Rit + AZT + 3TC
http://www.dsinfm.dk
•CD4 cell count < 200-300 cells/ul•Acute infection•Clinical Symptoms•Pregnancy
STATENS SERUM INSTITUT
Combination therapy:
Drugs Clinical effect Virological effect
HAART Highly Active Antiretroviral Therapy(3-4 drugsNRTI+NNRTI or PI)
STATENS SERUM INSTITUT
Combination therapy:
Drugs Clinical effect Virological effect
0
20000
40000
60000
80000
100000
120000
140000
160000
31-01-1993 15-06-1994 28-10-1995 11-03-1997 24-07-1998 06-12-1999
Vira
l load
Måneder
HAARTHAART
HAART Highly Active Antiretroviral Therapy(3-4 drugsNRTI+NNRTI or PI)
STATENS SERUM INSTITUT
Combination therapy:
Drugs Clinical effect Virological effect
0
20000
40000
60000
80000
100000
120000
140000
160000
31-01-1993 15-06-1994 28-10-1995 11-03-1997 24-07-1998 06-12-1999
Vira
l load
Måneder
HAARTHAART
HAART Highly Active Antiretroviral Therapy(3-4 drugsNRTI+NNRTI or PI)
STATENS SERUM INSTITUT
Combination therapy:
Drugs Clinical effect Virological effect
0
20000
40000
60000
80000
100000
120000
140000
160000
31-01-1993 15-06-1994 28-10-1995 11-03-1997 24-07-1998 06-12-1999
Vira
l load
Months
HAARTHAART
ComplianceSite-effectsDevelopment of resistance
HAART Highly Active Antiretroviral Therapy(3-4 drugsNRTI+NNRTI or PI)
STATENS SERUM INSTITUT
Compliance and Site-effects
STATENS SERUM INSTITUT
Mutations in the Pro and RT gene associated with resistance
Primary: D30N, M46I/L, G48V, I50V, V82F/A/T/S, I84V, L90M
Secondary: L10F/I/R/V, K20R/M, L24I/V, V32I, L33F, M36I, I47V, F53L, I54V/M/L, L63P, A71V/T, G73S/A, V77I, N88D/S
NRTI: M41L, E44D, A62V, K65R, D67N, T69D/(S+xx), K70R, L74V, V75T/M/A/I, F77L, Y115F, F116Y, V118I, Q151M, M184V/I, L210W, T215Y/F, K219Q/E
NNRTI: A98G, L100I, K101E, K103N, V106A/I, V108I, V179D, Y181C/I, Y188L/C/H, G190A/S, P225H, M230L, P236L
Development of resistance
STATENS SERUM INSTITUT
Time
Viral load
Fitness of the resistant virus
Speed of development of resistance
Effectivity of the drug against wild type
Development of resistant HIV against antiretroviral drugs
STATENS SERUM INSTITUT
Methods for genotyping
Commercial services:VircoVirologicetc.
Commercial kitsAbbott ViroSeqVisible Genetics
In House:Home brewed asays
STATENS SERUM INSTITUT
EDTA-blood sample
Extraction of HIV-RNA from plasma
cDNA syntesis by MuLV reverse transcriptase
Single PCR-amplification of 1,8 kb of the pol-gene
Direct sequencing of the protease- and RT-gene
Electroforesis (ABI 3100)
Nucleotide sequence
Translation to amino acidsequence
Identification of mutations associated to resistance90
10
54
84
82
71
63
20
32
46
24
4850
33
36
73
47
77
30
88
Genotypic resistance by direct sequencing
STATENS SERUM INSTITUT
Genotypic resistance testing on routine basis Genotypic resistance testing on routine basis in a quality insured (ISO 17025) laboratory: in a quality insured (ISO 17025) laboratory:
Department of Virology SSIDepartment of Virology SSI
EDTA-blood sample
Extraction of HIV-RNA from plasma
* Abbott ViroSeq 2 system
Nucleotide sequence
* Quality control by phylogenetic analysis
* Identification of mutations associated to resistance
* Interpretation of resistance
* Report
STATENS SERUM INSTITUT
STATENS SERUM INSTITUT
Genotypic resistance testing on routine basis Genotypic resistance testing on routine basis in a quality insured (ISO 17025) laboratory: in a quality insured (ISO 17025) laboratory:
Department of Virology SSIDepartment of Virology SSI
EDTA-blood sample
Extraction of HIV-RNA from plasma
* Abbott ViroSeq 2 system
Nucleotide sequence
* Quality control and subtyping by phylogenetic analysis
* Identification of mutations associated to resistance
* Interpretation of resistance
* Report
STATENS SERUM INSTITUT
Quality control by phylogenetic analysis
Nucleotide Substitutions (x100)0
12.2
24681012
pt1906PT1857G
pt1900pt1861
pt1908pt1911
pt1914gpt1889
pt1854pt1893
pt1895pt1901
pt1771pt1801
pt1262PT1915
PT1868pt1904B-hxb2
PT1864pt1912
PT1867pt1896
pt1903pt1852
pt1894gB-rf
pt1910D-eli
D-ndkpt1856
pt1858pt1880gg
pt1891C-BRO25
CH2220pt1909
pt1883gpt1897
pt1899F1-BR
F2MP255pt1902
pt1881AUG037pt1862
AETH253HCF056
GNG083pt1882g
JSE7022
Subtyping
eg. subtype D
STATENS SERUM INSTITUT
Genotypic resistance testing on routine basis Genotypic resistance testing on routine basis in a quality insured (ISO 17025) laboratory: in a quality insured (ISO 17025) laboratory:
Department of Virology SSIDepartment of Virology SSI
EDTA-blood sample
Extraction of HIV-RNA from plasma
* Abbott ViroSeq 2 system
Nucleotide sequence
* Quality control by phylogenetic analysis
* Identification of mutations associated to resistance
* Interpretation of resistance
* Report
STATENS SERUM INSTITUT
Identification of mutationer associated to resistance
STATENS SERUM INSTITUT
Genotypic resistance testing on routine basis Genotypic resistance testing on routine basis in a quality insured (ISO 17025) laboratory: in a quality insured (ISO 17025) laboratory:
Department of Virology SSIDepartment of Virology SSI
EDTA-blood sample
Extraction of HIV-RNA from plasma
* Abbott ViroSeq 2 system
Nucleotide sequence
* Quality control by phylogenetic analysis
* Identification of mutations associated to resistance
* Interpretation of resistance
* Report
STATENS SERUM INSTITUT
Interpretation of genotypic resistance
Tables www.iasusa.org
Rulebased algoritms www.hivdb.stanford.edu/hiv/
Virtual phenotypes www.virco.be
Neural networks
STATENS SERUM INSTITUT
Interpretation of genotypic resistanceDept. of Virology, SSI
STATENS SERUM INSTITUT
Genotypic resistance testing on routine basis Genotypic resistance testing on routine basis in a quality insured (ISO 17025) laboratory: in a quality insured (ISO 17025) laboratory:
Department of Virology SSIDepartment of Virology SSI
EDTA-blood sample
Extraction of HIV-RNA from plasma
* Abbott ViroSeq 2 system
Nucleotide sequence
* Quality control by phylogenetic analysis
* Identification of mutations associated to resistance
* Interpretation of resistance
* Report
STATENS SERUM INSTITUT
ReportDept. of Virology, SSI
STATENS SERUM INSTITUT
New phenotypic assay:Recombinant Virus Assay (RVA)
Recombinant virus
MTT-assay(IC-50)
STATENS SERUM INSTITUT
Cross-resistance
Susceptible Genotypic WT (K103)
Phenotypic Fold = 1
J.Virology 2001, 75(11)
STATENS SERUM INSTITUT
Cross-resistance
Susceptible K103N Genotypic WT (K103)
Resistant
Phenotypic Fold = 1 Fold = 57-437X
J.Virology 2001, 75(11)
STATENS SERUM INSTITUT
0
100
200
300
400
500
600
31-01-93 15-06-94 28-10-95 11-03-97 24-07-98 06-12-99
CD
4 c
ou
nt
0
50000
100000
150000
200000
250000
300000
350000
Vir
al
loa
d
AZT3TC SAQ ddC
D4TCRX NOR
ddINEVNEL EFA
ABCHYD
M184VM41LD67NK70R
L210WT215YK219EG190AY181C
L10IM46IL63PA71VV77II84V
L90M
M184VM41LD67NK70R
L210WT215YK219EL74I
Y181C
L10IM46IL63PL90M
0.1
1.0
10.0
100.0
1000.0NEV
ABC
AZT
3TC
D4T
CRX
NOR
SAQ
0.1
1.0
10.0
100.0
1000.0NEV
ABC
D4T
AZT
3TCNEL
CRX
NOR
SAQ
STATENS SERUM INSTITUT
Viral load
0
50
100
150
200
250
300
350ja
n-9
6
jul-
96
jan
-97
jul-
97
jan
-98
jul-
98
jan
-99
jul-
99
jan
-00
jul-
00
jan
-01
jul-
01
jan
-02
Viral load
AZT3TCd4TABCddINevIndNelRit
RNA/DNA
TI-2 start
(x 1000 kopier/ml)
RNA/DNA
W184,W215
W184,M215W184,R215
M184,R215
M184,M215
W184,M215
M=mutantW=wildtypeR=revertant
W184,M215
M184,M215
TI-1 start
M184,M215(jul 2002)
Resumed treatment
W184,W215
STATENS SERUM INSTITUT120194
281191
300502D
150502D
150502
150296
300502
250597*
201097*
170698*
070499*
121200
030701*
180702*
170102*
201099
201099D
ASE7253
AUG037
Treatment interuption 1
Treatment interuption 2
T215YT215C/S*M184V
97
56
61
29
Fylogeni af Pol-genet
STATENS SERUM INSTITUT
Detection limit of Genotypic resistance assay
STATENS SERUM INSTITUT
0
1000
2000
3000
4000
5000
6000
17
.03
.97
16
.04
.97
28
.07
.97
24
.09
.97
29
.12
.97
01
.04
.98
02
.07
.98
08
.09
.98
07
.12
.98
01
.03
.99
07
.06
.99
07
.09
.99
01
.11
.99
07
.02
.00
Copies/ml
Protease
RT
L63PV77I
L74VM184V
New ultra sensitive assay for genotypic resistance
STATENS SERUM INSTITUT
0
1000
2000
3000
4000
5000
6000
17
.03
.97
16
.04
.97
28
.07
.97
24
.09
.97
29
.12
.97
01
.04
.98
02
.07
.98
08
.09
.98
07
.12
.98
01
.03
.99
07
.06
.99
07
.09
.99
01
.11
.99
07
.02
.00
Copies/ml
Protease
RT
L63PV77I
L74VM184V
Improved extractionNested / double PCR=> 20 copies pr ml
New ultra sensitive assay for genotypic resistance
STATENS SERUM INSTITUT
0
1000
2000
3000
4000
5000
6000
17
.03
.97
16
.04
.97
28
.07
.97
24
.09
.97
29
.12
.97
01
.04
.98
02
.07
.98
08
.09
.98
07
.12
.98
01
.03
.99
07
.06
.99
07
.09
.99
01
.11
.99
07
.02
.00
Copies/ml
Protease
RT
L63PV77I
L74VM184V
L74VM184V
L63PV77I
M36IL63PV77IN88DL90M*
L33FM36IL63PV77II84VN88DL90M*
L33FM36IL63PV77II84VN88D
L33FM36IL63PV77II84VN88D
L33FM36IL63PV77II84VN88DL90M*
M36IL63PV77II84VL90M*
L33FM36IL63PV77II84VN88DL90M*
K20TL24ID30N*M36IL63PV77IN88DL90M*
Improved extractionNested / double PCR=> 20 copies pr ml
New ultra sensitive assay for genotypic resistance
STATENS SERUM INSTITUT
Therapy
Development ofDrug resistance
STATENS SERUM INSTITUT
Therapy
Development of Drug resistance
Transmission of resistance
STATENS SERUM INSTITUT
Transmission of resistant HIV-1 strains
RTI:PI:
Spain 3% 1% Italy 11% 2% Belgium 13% 4% Luxembourg 12% 0% Germany 16% 3% France 17% 2%
Europe
Total MDR: Primary HIV:
1995-1998 8% 4%
1999-2000 23% 10%
RTI: PI: Seroconv. 6% 1% IDVU 0% 0% New diagn. 13% 3% Military 10% 10%
USA
STATENS SERUM INSTITUT
Reversion of genotypic resistance
AZT 215
ACC(T)
WT
A. DeRonde J.Virol.2001
0 1 2 År
STATENS SERUM INSTITUT
Reversion of genotypic resistance
AZT 215
TAC(Y)ACC(T)
WT
A. DeRonde J.Virol.2001
0 1 2 År
STOP of therapy
STATENS SERUM INSTITUT
Reversion of genotypic resistance
AZT 215
TCC(S)
GAC(D)
TAC(Y)ACC(T) AAC(N)
WT
A. DeRonde J.Virol.2001
0 1 2 År
STOP of therapy
STATENS SERUM INSTITUT
Reversion of genotypic resistance
AZT 215
TCC(S)
GAC(D) AGC(S)
TAC(Y)ACC(T) AAC(N) GAC(D)
WT
A. DeRonde J.Virol.2001
0 1 2 År
STOP of therapy
STATENS SERUM INSTITUT
2000: 2,0% (2/104) 41L, 210S, 215S 215S
2001: 4,8% (7/147) 90M, 63P, 71V, 77I 215E
215L (x2) 215S 215S 82F, 10I, 63P
98G, 215S
2002: 5,0% (7/140) 103N, 215Y 103N, 188C
41L, 215D 90M, 10I, 63P, 71T, 77I215D (x2)215S67N
2003: 5,5% (7/127) 67N, 219Q 67N, 219E 84V, 10V,33F,36I,63P
69D 215N215S103N181C
2004: 0,7% (1/137) 190A
Total prv. RT PI
Prevalence of drug resistance mutations in newly diagnosed HIV-1 patients in Denmark 2000-2004
STATENS SERUM INSTITUT
HIV-1 subtypes
Based on the variation ofthe genome HIV can be divided in to different subtypes
D
CF
E
O
B
G
A
H
STATENS SERUM INSTITUT
HIV-1 subtypes
9 identified subtypes (A-K) and 16 CRF (circulating recombinantforms)
In the industrialised world subtype B is mostprevalent. However, globally subtype C is the most prevalent subtype.
STATENS SERUM INSTITUT
Prevalence of subtype non-B in newly diagnosed HIV-1 patients in Denmark
Subtype 2000
A8%
AE4% AG
5%
C18%
D3%
G2%
B59%
Subtype 2001
A9% AE
7% AG3%
B63%
C12%
D2%
G1%
cpx3%
30%
12%47%
5% 6%
Global subtype distribution
C
DAndre
A
B
Ref.: J.Clin. Virol. 29 (2004)
STATENS SERUM INSTITUT
EpidemilogyTransmission route of infection 2001
0%
10%
20%
30%
40%
50%
2001-SUR 33% 47% 9% 7%
2001 36% 46% 10% 8%
MSM HTX IVDU Unknown
Subtype 2001
A9% AE
7% AG3%
B63%
C12%
D2%
G1%
cpx3%
Country of infection 2001
Denmark51%
Europe2%
Afrika22%
Asia8%
NorthAmerika1%
Unknown16%
STATENS SERUM INSTITUT
2000: 2,0% (2/104) 41L, 210S, 215S B 41 % 0/2 215S B
2001: 4,8% (7/147) 90M, 63P, 71V, 77I B 39 % 0/7 215E B
215L (x2) B+B 215S B 215S 82F, 10I, 63P B
98G, 215S B
2002: 5,0% (7/140) 103N, 215Y B 37 % 4/7 103N, 188C AG
41L, 215D 90M, 10I, 63P, 71T, 77I B215D (x2) B+D215S AE67N A
2003: 5,5% (7/127) 67N, 219Q B 34 % 4/7 67N, 219E 84V, 10V,33F,36I,63P AE
69D AE215N B215S B103N A181C AE
2004: 0,7% (1/137) 190A AD (34 %) 0/1
Total prv. RT PI Subtype % non-B Prv. Non-B
Prevalence of drug resistance mutations in newly diagnosed HIV-1 patients in Denmark 2000-2004
STATENS SERUM INSTITUT
Transmission of HIV
STATENS SERUM INSTITUT
Case
African/danish family African mother - HIV pos in 2004 African daugter – HIV pos in 2005
– Probably infected at birth – Arrived as teenager in DK– Has no HIV symptoms
Danish stepfather – HIV positiv 2003– Infected by african wife
STATENS SERUM INSTITUT
Questions:
Can we confirm the mother has infected the daughter?
Is it possible to identify af transmission chain after 17 years?
STATENS SERUM INSTITUT
Nucleotide Substitutions (x100)0
20.0
24681012141618
sero386A1.KE.00.MSA4076
A1.KE.00.KER2009SERO630
A1.KE.00.KNH1211A1.KE.99.KSM4021
A1.KE.00.MSA4069A1.KE.00.KNH1209sero736
A1.KE.00.KNH1199sero531g
sero913zse1520
se762se1712A1.KE.00.KNH1144
A1.KE.00.KER2008A1.KE.94.Q23_17
A1.KE.99.KNH1135A1.KE.97.ML605_3_1997
se1071A1.KE.00.MSA4079A1.KE.97.ML752se1033
se983vsero553
A1.KE.86.ML013_10_1986A1.KE.97.ML013_2_1997
sero350ggA1.KE.00.KSM4024
sero434sero838
sero369AUG037A1.KE.00.NKU3005
sero394A1.KE.00.KER2012
A1.KE.99.KNH1088A1.KE.00.MSA4070
A1.KE.86.ML170_1986sero530gsero540
SERO705se1084
sero801sero904
sero379sero519
sero655sero411p
sero560A1.KE.00.KNH1207
SERO670UGA1.KE.00.MSA4072
A1.KE.00.KSM4030sero558
sero1035Sero360
se1037SERO744UG
SERO1122SERO878
AETH253sero454
sero943PatientrefB-hxb2
B-rfD-eli
D-ndkF1-BR
F2MP255GNG083
JSE7022HCF056C-BRO25CH2220
SE1501(se1498)Omvp5180
Nucleotide Substitutions (x100)0
20.0
24681012141618
sero386A1.KE.00.MSA4076
A1.KE.00.KER2009SERO630
A1.KE.00.KNH1211A1.KE.99.KSM4021
A1.KE.00.MSA4069A1.KE.00.KNH1209sero736
A1.KE.00.KNH1199sero531g
sero913zse1520
se762se1712A1.KE.00.KNH1144
A1.KE.00.KER2008A1.KE.94.Q23_17
A1.KE.99.KNH1135A1.KE.97.ML605_3_1997
se1071A1.KE.00.MSA4079A1.KE.97.ML752se1033
se983vsero553
A1.KE.86.ML013_10_1986A1.KE.97.ML013_2_1997
sero350ggA1.KE.00.KSM4024
sero434sero838
sero369AUG037A1.KE.00.NKU3005
sero394A1.KE.00.KER2012
A1.KE.99.KNH1088A1.KE.00.MSA4070
A1.KE.86.ML170_1986sero530gsero540
SERO705se1084
sero801sero904
sero379sero519
sero655sero411p
sero560A1.KE.00.KNH1207
SERO670UGA1.KE.00.MSA4072
A1.KE.00.KSM4030sero558
sero1035Sero360
se1037SERO744UG
SERO1122SERO878
AETH253sero454
sero943PatientrefB-hxb2
B-rfD-eli
D-ndkF1-BR
F2MP255GNG083
JSE7022HCF056C-BRO25CH2220
SE1501(se1498)Omvp5180
94100
78 (Daughter)
(Mother)
(Stepfather)
POL
Metode: Neighbour joining tree, 100 bootstrap replikations 1200 bp All HIV-1 subtype A POL sequences from Los Alamos included Subtype A sequences from SERO project
Bootstrapvalues over 50% is shown
STATENS SERUM INSTITUT
Nucleotide Substitutions (x100)0
18.1
24681012141618
AY772968AY772969
AY772985AY322184
AY322185L11774AF159877
L11772AF095833
AY322193L11775
L11757AF159878
AY772954L11786
L11794AF361873U56356
se762se1520
se1712AY772970
L11773AY322190AF095832AY772958
AY772955L11770
AF539405L11768
AB098332AB098333
U56359AY772953
L11788L11791AF004885
U56360AF159876
AF481779AB098330AB098331
AY772971U51190
AF481778AY772986
AY187680AY456310
AY676592AY676591
AY676593AY676594
AY456312AY456313AY456314
AY456316AY456315
L11798M62320
L11777AY772973
AY772975AY772983
AY772974AY772976
AY772978K03455
AB081213AY456330
AF286238L11783
Nucleotide Substitutions (x100)0
18.1
24681012141618
AY772968AY772969
AY772985AY322184
AY322185L11774AF159877
L11772AF095833
AY322193L11775
L11757AF159878
AY772954L11786
L11794AF361873U56356
se762se1520
se1712AY772970
L11773AY322190AF095832AY772958
AY772955L11770
AF539405L11768
AB098332AB098333
U56359AY772953
L11788L11791AF004885
U56360AF159876
AF481779AB098330AB098331
AY772971U51190
AF481778AY772986
AY187680AY456310
AY676592AY676591
AY676593AY676594
AY456312AY456313AY456314
AY456316AY456315
L11798M62320
L11777AY772973
AY772975AY772983
AY772974AY772976
AY772978K03455
AB081213AY456330
AF286238L11783
10068
StepfatherMother
Daughter
Gag
Metode: Neighbour joining tree, 100 bootstrap replikations 370 bp All HIV-1 subtype A gag sequences from Los Alamos included Bootstrapvalues over 50% is shown
STATENS SERUM INSTITUT
GAG
Daughter!
STATENS SERUM INSTITUT
Dublication
GAG
STATENS SERUM INSTITUT
Epidemiological Study of HIV-1 in Greenland using Phylogenetic Analyses
TV Madsen, N Obel, N Lohse, J Gerstoft, AB Petersen, C Nielsen & LB Jørgensen
•90 samples from Greenland HIV-1 positive patients with high viral load, and as early in the infection as possible, was chosen for the study. HIV-1 sequences from Danish patients are also included in order to compare the genetic composition of the two populations.
•Sequences were obtained from selected regions (gag: p17-region; pol: protease and 250 aa of RT; env: V3-region) using in-house PCR and sequencing methods, and ViroSeqTM HIV Genotyping System (Abbott).
•In order to study the genetic variation over time and to identify local outbreaks and chains of infection in the Greenland HIV-1 population the phylogeny was correlated with epidemiological data from the Danish HIV Cohort.
STATENS SERUM INSTITUT
Figure 1. Phylogeny on pol-region (NJ, 100 bootstrap replicates). Isolates from Greenland have been marked in green.
0.1
SE297 ALB.US.83.RF
SE484 HHB.US.90.WE
SE123 KHSE476 RH
SE180 RHGR46 DIH
GR83 QASSE161 HH
BIO28 KHSE118 KH
SE212 MASE93 KHSE174 RH
SE237 RHGR72 DIH
SE191 RHSE520 RH
SE348 MAJ.SE.93.SE
J.SE.94.SE13 CPX.CM.
11 CPX.FR.11 CPX.CM.11 CPX.GR.
SE402 RHSE214 RH
SE178 AL01 AE.CF.9
GR1 UPS01 AE.TH.9
SE138 RHSE140 KH
SE176 RHSE417 RH
GR25 DIHGR73 DIH
GR3 DIHGR4 DIH
GR58 DIHGR38 DIHGR67 DIH
GR71 DIHGR63 DIH
GR52 DIHGR28 DIHGR53 DIH
GR42 DIHGR43 DIH
GR26 DIHGR27 DIH
GR7 DIHGR47 MANS
GR23 DIHGR65 DIH
GR57 DIHGR41 SSHGR75 DIH
GR14 SSHGR55 SSH
GR62 SSHGR70 SSH
GR9 SSHGR11 SSH
GR44 SSHGR17 SSH
GR40 ASSGR12 SSH
GR82 SSHGR19 SSHGR92 SSH
GR78 SSHGR34 SSH
GR30 SSHGR15 SSHGR76 SSH
B.FR.83.HXSE112 RH
ddI-23 RHSE263 RH
GR22 ISB.US.86.JR
SE181 RHSE136 KH
SE148 RHGR2 DIH
GR54 DIHGR56 DIH
SE131 KHSE249 AL
SE199 RHSE134 RH
SE100 RHSE175 RH
BIO17 KHSE203 RH
93
10080
100
71
100
83
81
83
100100
100
99100
100
0.1
SE297 ALB.US.83.RF
SE484 HHB.US.90.WE
SE123 KHSE476 RH
SE180 RHGR46 DIH
GR83 QASSE161 HH
BIO28 KHSE118 KH
SE212 MASE93 KHSE174 RH
SE237 RHGR72 DIH
SE191 RHSE520 RH
SE348 MAJ.SE.93.SE
J.SE.94.SE13 CPX.CM.
11 CPX.FR.11 CPX.CM.11 CPX.GR.
SE402 RHSE214 RH
SE178 AL01 AE.CF.9
GR1 UPS01 AE.TH.9
SE138 RHSE140 KH
SE176 RHSE417 RH
GR25 DIHGR73 DIH
GR3 DIHGR4 DIH
GR58 DIHGR38 DIHGR67 DIH
GR71 DIHGR63 DIH
GR52 DIHGR28 DIHGR53 DIH
GR42 DIHGR43 DIH
GR26 DIHGR27 DIH
GR7 DIHGR47 MANS
GR23 DIHGR65 DIH
GR57 DIHGR41 SSHGR75 DIH
GR14 SSHGR55 SSH
GR62 SSHGR70 SSH
GR9 SSHGR11 SSH
GR44 SSHGR17 SSH
GR40 ASSGR12 SSH
GR82 SSHGR19 SSHGR92 SSH
GR78 SSHGR34 SSH
GR30 SSHGR15 SSHGR76 SSH
B.FR.83.HXSE112 RH
ddI-23 RHSE263 RH
GR22 ISB.US.86.JR
SE181 RHSE136 KH
SE148 RHGR2 DIH
GR54 DIHGR56 DIH
SE131 KHSE249 AL
SE199 RHSE134 RH
SE100 RHSE175 RH
BIO17 KHSE203 RH
0.1
SE297 ALB.US.83.RF
SE484 HHB.US.90.WE
SE123 KHSE476 RH
SE180 RHGR46 DIH
GR83 QASSE161 HH
BIO28 KHSE118 KH
SE212 MASE93 KHSE174 RH
SE237 RHGR72 DIH
SE191 RHSE520 RH
SE348 MAJ.SE.93.SE
J.SE.94.SE13 CPX.CM.
11 CPX.FR.11 CPX.CM.11 CPX.GR.
SE402 RHSE214 RH
SE178 AL01 AE.CF.9
GR1 UPS01 AE.TH.9
SE138 RHSE140 KH
SE176 RHSE417 RH
GR25 DIHGR73 DIH
GR3 DIHGR4 DIH
GR58 DIHGR38 DIHGR67 DIH
GR71 DIHGR63 DIH
GR52 DIHGR28 DIHGR53 DIH
GR42 DIHGR43 DIH
GR26 DIHGR27 DIH
GR7 DIHGR47 MANS
GR23 DIHGR65 DIH
GR57 DIHGR41 SSHGR75 DIH
0.1
SE297 ALB.US.83.RF
SE484 HHB.US.90.WE
SE123 KHSE476 RH
SE180 RHGR46 DIH
GR83 QASSE161 HH
BIO28 KHSE118 KH
SE212 MASE93 KHSE174 RH
SE237 RHGR72 DIH
SE191 RHSE520 RH
SE348 MAJ.SE.93.SE
J.SE.94.SE13 CPX.CM.
11 CPX.FR.11 CPX.CM.11 CPX.GR.
SE402 RHSE214 RH
SE178 AL01 AE.CF.9
GR1 UPS01 AE.TH.9
SE138 RHSE140 KH
SE176 RHSE417 RH
GR25 DIHGR73 DIH
GR3 DIHGR4 DIH
GR58 DIHGR38 DIHGR67 DIH
GR71 DIHGR63 DIH
GR52 DIHGR28 DIHGR53 DIH
GR42 DIHGR43 DIH
GR26 DIHGR27 DIH
GR7 DIHGR47 MANS
GR23 DIHGR65 DIH
GR57 DIHGR41 SSHGR75 DIH
GR14 SSHGR55 SSH
GR62 SSHGR70 SSH
GR9 SSHGR11 SSH
GR44 SSHGR17 SSH
GR40 ASSGR12 SSH
GR82 SSHGR19 SSHGR92 SSH
GR78 SSHGR34 SSH
GR30 SSHGR15 SSHGR76 SSH
B.FR.83.HXSE112 RH
ddI-23 RHSE263 RH
GR22 ISB.US.86.JR
SE181 RHSE136 KH
SE148 RHGR2 DIH
GR54 DIHGR56 DIH
SE131 KHSE249 AL
SE199 RHSE134 RH
SE100 RHSE175 RH
BIO17 KHSE203 RH
93
10080
100
71
100
83
81
83
100100
100
99100
100
9774
8872
98
91
STATENS SERUM INSTITUT
Figure 1. Phylogeny on pol-region (NJ, 100 bootstrap replicates). Isolates from Greenland have been marked in green.
0.1
SE297 ALB.US.83.RF
SE484 HHB.US.90.WE
SE123 KHSE476 RH
SE180 RHGR46 DIH
GR83 QASSE161 HH
BIO28 KHSE118 KH
SE212 MASE93 KHSE174 RH
SE237 RHGR72 DIH
SE191 RHSE520 RH
SE348 MAJ.SE.93.SE
J.SE.94.SE13 CPX.CM.
11 CPX.FR.11 CPX.CM.11 CPX.GR.
SE402 RHSE214 RH
SE178 AL01 AE.CF.9
GR1 UPS01 AE.TH.9
SE138 RHSE140 KH
SE176 RHSE417 RH
GR25 DIHGR73 DIH
GR3 DIHGR4 DIH
GR58 DIHGR38 DIHGR67 DIH
GR71 DIHGR63 DIH
GR52 DIHGR28 DIHGR53 DIH
GR42 DIHGR43 DIH
GR26 DIHGR27 DIH
GR7 DIHGR47 MANS
GR23 DIHGR65 DIH
GR57 DIHGR41 SSHGR75 DIH
GR14 SSHGR55 SSH
GR62 SSHGR70 SSH
GR9 SSHGR11 SSH
GR44 SSHGR17 SSH
GR40 ASSGR12 SSH
GR82 SSHGR19 SSHGR92 SSH
GR78 SSHGR34 SSH
GR30 SSHGR15 SSHGR76 SSH
B.FR.83.HXSE112 RH
ddI-23 RHSE263 RH
GR22 ISB.US.86.JR
SE181 RHSE136 KH
SE148 RHGR2 DIH
GR54 DIHGR56 DIH
SE131 KHSE249 AL
SE199 RHSE134 RH
SE100 RHSE175 RH
BIO17 KHSE203 RH
93
10080
100
71
100
83
81
83
100100
100
99100
100
0.1
SE297 ALB.US.83.RF
SE484 HHB.US.90.WE
SE123 KHSE476 RH
SE180 RHGR46 DIH
GR83 QASSE161 HH
BIO28 KHSE118 KH
SE212 MASE93 KHSE174 RH
SE237 RHGR72 DIH
SE191 RHSE520 RH
SE348 MAJ.SE.93.SE
J.SE.94.SE13 CPX.CM.
11 CPX.FR.11 CPX.CM.11 CPX.GR.
SE402 RHSE214 RH
SE178 AL01 AE.CF.9
GR1 UPS01 AE.TH.9
SE138 RHSE140 KH
SE176 RHSE417 RH
GR25 DIHGR73 DIH
GR3 DIHGR4 DIH
GR58 DIHGR38 DIHGR67 DIH
GR71 DIHGR63 DIH
GR52 DIHGR28 DIHGR53 DIH
GR42 DIHGR43 DIH
GR26 DIHGR27 DIH
GR7 DIHGR47 MANS
GR23 DIHGR65 DIH
GR57 DIHGR41 SSHGR75 DIH
GR14 SSHGR55 SSH
GR62 SSHGR70 SSH
GR9 SSHGR11 SSH
GR44 SSHGR17 SSH
GR40 ASSGR12 SSH
GR82 SSHGR19 SSHGR92 SSH
GR78 SSHGR34 SSH
GR30 SSHGR15 SSHGR76 SSH
B.FR.83.HXSE112 RH
ddI-23 RHSE263 RH
GR22 ISB.US.86.JR
SE181 RHSE136 KH
SE148 RHGR2 DIH
GR54 DIHGR56 DIH
SE131 KHSE249 AL
SE199 RHSE134 RH
SE100 RHSE175 RH
BIO17 KHSE203 RH
0.1
SE297 ALB.US.83.RF
SE484 HHB.US.90.WE
SE123 KHSE476 RH
SE180 RHGR46 DIH
GR83 QASSE161 HH
BIO28 KHSE118 KH
SE212 MASE93 KHSE174 RH
SE237 RHGR72 DIH
SE191 RHSE520 RH
SE348 MAJ.SE.93.SE
J.SE.94.SE13 CPX.CM.
11 CPX.FR.11 CPX.CM.11 CPX.GR.
SE402 RHSE214 RH
SE178 AL01 AE.CF.9
GR1 UPS01 AE.TH.9
SE138 RHSE140 KH
SE176 RHSE417 RH
GR25 DIHGR73 DIH
GR3 DIHGR4 DIH
GR58 DIHGR38 DIHGR67 DIH
GR71 DIHGR63 DIH
GR52 DIHGR28 DIHGR53 DIH
GR42 DIHGR43 DIH
GR26 DIHGR27 DIH
GR7 DIHGR47 MANS
GR23 DIHGR65 DIH
GR57 DIHGR41 SSHGR75 DIH
0.1
SE297 ALB.US.83.RF
SE484 HHB.US.90.WE
SE123 KHSE476 RH
SE180 RHGR46 DIH
GR83 QASSE161 HH
BIO28 KHSE118 KH
SE212 MASE93 KHSE174 RH
SE237 RHGR72 DIH
SE191 RHSE520 RH
SE348 MAJ.SE.93.SE
J.SE.94.SE13 CPX.CM.
11 CPX.FR.11 CPX.CM.11 CPX.GR.
SE402 RHSE214 RH
SE178 AL01 AE.CF.9
GR1 UPS01 AE.TH.9
SE138 RHSE140 KH
SE176 RHSE417 RH
GR25 DIHGR73 DIH
GR3 DIHGR4 DIH
GR58 DIHGR38 DIHGR67 DIH
GR71 DIHGR63 DIH
GR52 DIHGR28 DIHGR53 DIH
GR42 DIHGR43 DIH
GR26 DIHGR27 DIH
GR7 DIHGR47 MANS
GR23 DIHGR65 DIH
GR57 DIHGR41 SSHGR75 DIH
GR14 SSHGR55 SSH
GR62 SSHGR70 SSH
GR9 SSHGR11 SSH
GR44 SSHGR17 SSH
GR40 ASSGR12 SSH
GR82 SSHGR19 SSHGR92 SSH
GR78 SSHGR34 SSH
GR30 SSHGR15 SSHGR76 SSH
B.FR.83.HXSE112 RH
ddI-23 RHSE263 RH
GR22 ISB.US.86.JR
SE181 RHSE136 KH
SE148 RHGR2 DIH
GR54 DIHGR56 DIH
SE131 KHSE249 AL
SE199 RHSE134 RH
SE100 RHSE175 RH
BIO17 KHSE203 RH
93
10080
100
71
100
83
81
83
100100
100
99100
100
9774
8872
98
91
?
STATENS SERUM INSTITUT
Transmission
1991
1994
2001
STATENS SERUM INSTITUT
Three patients:
•GR41: 31 year old female HTX infected in Greenland first positive test is from 27/9-94. Analysed sample from 14/7-99.•GR57: 51 year old male HTX infected in Greenland first
positive test 1/1-91. † 6/3-01. Analysed sample from 9/2-00.•GR75: 52 year old female HTX infected in Greenland first positive test is from 1/8-01. Analysed sample from 14/-02.
STATENS SERUM INSTITUT
Transmission
1991
1994
2001
9/2-2000
14/7-1999
14/1-2002
Analysed
STATENS SERUM INSTITUT
New mutation against NRTI?
STATENS SERUM INSTITUT
History
•Heavily treated patient with non-compliant behaviour
•Four samples sent for HIV-1 genotypic resistance
STATENS SERUM INSTITUT
Jun 2000Jan 2002Jan 2003Mar 2005
Mutation D67G_Y
STATENS SERUM INSTITUT
STATENS SERUM INSTITUTRegalInst algoritme
STATENS SERUM INSTITUT
juli 2000 januar 2002 januar 2003 marts 2005
D67N N N G G
D67GY GY GY
T69N N
K70R R R R
L74V V
V118I I
M184V V
K219Q Q Q Q
K103N KN
V108I I
Y181C C C C
M46I MI
V82A A A A
L90M M M M
L10I I I I
K20R R
M36I I I I
F53L L
I54V V V
L63P P P P
A71V V
V77I I
NRTI NNRTI PI
1998 ddi+abc nvp idv
Apr. 2003 (abc+3tc+azt)+tf rtv+amp
Jun. 2003 (abc+3tc+azt)+tf rtv+saq
Mar. 2005 (abc+3tc+azt)+tf rtv+saq
HIV drug mutations 2000-2005
STATENS SERUM INSTITUT
Treatment history
NRTI NNRTI PI VL Genotypic Resistance
1990-1998 AZT+ddI
1995-dec.1996 AZT+3TC
Dec.1996-1998 d4T+3TC IDV
1998 DDI+ABC NVP NFV
1998 DDI+ABC NVP IDV
juli 2000 DDI+ABC NVP IDV 54100 No insert: D67GY
Jan. 2002 DDI+ABC NVP IDV 56300 No insert: D67GY
Jan. 2003 DDI+ABC NVP IDV 40000 Plus insert: D67GY
Apr. 2003 (Abc+3TC+AZT)+TF RTV+Amp
Jun. 2003 (Abc+3TC+AZT)+TF RTV+SAQ
marts 2005 (Abc+3TC+AZT)+TF RTV+SAQ 16300 Plus insert: D67GY
STATENS SERUM INSTITUT
Virus BL-3 laboratory, Department of Virology, SSI
Deptartment of infectious diseasesRigshospitaletHvidovre HospitalSkejby SygehusOdense Universitets HospitalÅlborg SygehusSygehus Øresund
SupportABI and Abbott
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