A Robust Titanium Isophthalate Metal-Organic Framework for Visible-Light Photocatalytic CO2 Methanation

Full text of DOI:10.1016/j.chempr.2020.10.017

International Journal of Infectious Diseases 15 (2011) e722–e726
Contents lists available at ScienceDirect
International Journal of Infectious Diseases
journal homepage: www.elsevier.com/locate/ijid
Antiviral drug-associated potential vaccine-escape hepatitis B virus mutants in
Turkish patients with chronic hepatitis B
a,
b
M. Sayan *, S.C. Akhan
a
Clinical Laboratory, PCR Unit, Faculty of Medicine, University of Kocaeli, 41380 I˙zmit-Kocaeli, Turkey
b
Department of Clinical Bacteriology and Infectious Diseases, Faculty of Medicine, University of Kocaeli, Kocaeli, Turkey
A R T I C L E I N F O
S U M M A R Y
Article history:
Background: The hepatitis B virus (HBV) polymerase (pol) gene completely overlaps with the envelope (S)
gene. Mutations in the pol gene of HBV, either from selection of primary or secondary resistance
mutations, typically result in changes in the overlapping hepatitis B surface antigen (HBsAg). Recent
studies have conferred a new acronym to these HBV pol/S gene overlap mutants: ADAPVEMs, for antiviral
drug-associated potential vaccine-escape mutants. The present study aimed to assess the prevalence and
pattern of ADAPVEMs in Turkish patients with chronic hepatitis B (CHB).
Received 17 January 2011
Received in revised form 13 May 2011
Accepted 30 May 2011
Corresponding Editor: Mark Holodniy,
California, USA
Methods: The investigation was conducted between March 2007 and July 2010 and involved a total of
442 patients. These patients were in the following phases of HBV infection: immune tolerant (n = 50),
immune reactive (n = 37), inactive carrier (n = 90), HBeAg-negative CHB (n = 217), and HBsAg-negative
Keywords:
Hepatitis B virus
Antiviral agents
HBV polymerase
HBsAg
(
n = 12), or were hemodialysis patients (n = 36). One hundred eighty-six patients were receiving
nucleos(t)ide analogue (NUC) therapy and 256 patients had treatment-na ı¨ ve CHB.
Results: Seven types of ADAPVEM were detected in the total CHB patients: rtM204 V/sI195 M, rtM204I/
sW196S, rtM204I/sW196L, rtV173L/sE164D, rtA181T/sW172*, rtA181T/sW172L, and rtA181 V/sL173F.
The ADAPVEMs were associated with lamivudine, telbivudine, and adefovir. The prevalence of
ADAPVEMs in all CHB patients was found to be 10% (46/442). The difference in the prevalence of
ADAPVEMs across the different CHB clinical phases was not significant (Pearson Chi-square, p = 0.112).
The prevalence of ADAPVEMs was 24% (44/186) in those undergoing NUC therapy and 0.7% (2/256) in the
treatment-na ı¨ ve group; this difference was significant (Pearson Chi-square, p = 0.00).
Conclusions: We determined the prevalence and pattern of ADAPVEMs in Turkish patients in the
different phases of CHB. Preferred drugs in Turkey, such as lamivudine, have the potential to cause the
emergence of ADAPVEMs, with the possibility that these will spread to both individuals immunized with
the hepatitis B vaccine and nonimmunized individuals. ADAPVEMs should be monitored in infected and
treated patients and their public health risks assessed.
ß 2011 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
into two groups: those that cause resistance (primary), which
sometimes lead to a decreased viral fitness, and compensatory
1
. Introduction
(
secondary) mutations, which partially or fully restore the level of
In many parts of the world, five hepatitis B virus (HBV)-specific
3
viral fitness. In addition, it is important to keep in mind that the
HBV polymerase (pol) gene completely overlaps with the envelope
(
either from selection of primary or secondary resistance muta-
tions, typically result in changes in the overlapping hepatitis B
surface antigen (HBsAg). The results of changes are firstly that HBV
replication may be restored by these pol gene mutations and result
in relapse of hepatitis B infection with severe liver disease, and
secondly that there may be a failure of infection prevention with
nucleos(t)ide analogues (NUCs) in three subclasses targeting the
viral polymerase are approved for the treatment of chronic
hepatitis B (CHB): L-nucleosides (lamivudine, LAM; telbivudine,
LdT), deoxyguanosine analogues (entecavir, ETV), and acyclic
4
S) gene. Therefore, mutations in the pol open reading frame,
1
nucleoside phosphonates (adefovir, ADV; tenofovir, TDF).
A major concern with NUC treatment is the selection of
antiviral-resistant mutations. In particular, long-term therapy
with NUCs is associated with an increasing risk of the development
2
of drug resistance. Mutations selected under NUCs can be split
3
vaccination or hepatitis B immunoglobulin (HBIg).
LAM selected mutants – sE164D, sM198I, sI195 M, and sW196S
–
have shown reduced binding to hepatitis B surface antibody
*
Corresponding author. Tel.: +90 262 303 8571; fax: +90 262 303 8085.
3
–5
E-mail address: sayanmurat@hotmail.com (M. Sayan).
(anti-HBs).
Likewise, the point mutation that causes the
1
201-9712/$36.00 – see front matter ß 2011 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.ijid.2011.05.019

M. Sayan, S.C. Akhan / International Journal of Infectious Diseases 15 (2011) e722–e726
e723
rtA181T change in the pol also encodes a stop codon (sW172*) in
HBsAg. One recent report has provided evidence of involvement of
245 mg/day; Gilead Sciences, Inc.) as monotherapy or in combina-
tion therapy. All of the patients were categorized as chronic HBV
carriers according to the European Association for the Study of the
6
HBV encoding the rtA181T/sW172* mutation in the pathogenesis
7
1
of, and progression to hepatocellular carcinoma (HCC). Some of
Liver (EASL) clinical practice guidelines. The timing of serum
the corresponding changes in HBsAg are associated with resistance
sample collection was based on virological and/or biochemical
breakthrough in those patients who were receiving NUC therapy.
Blood samples were immediately separated by centrifugation, and
the serum separated, aliquoted, and kept at ꢀ20 8C until testing.
Serological markers of HBV (HBsAg, anti-HBs, antibodies to hepatitis
B core antigen (anti-HBc), hepatitis B e antigen (HBeAg), and
antibody to hepatitis B e antigen (anti-HBe)) were tested using
commercially available microparticle enzyme immunoassay kits
(Axsym, Abbott Laboratories, IL, USA and Elecsys, Roche Diagnostics,
Mannheim, Germany). However, serological data showed that all
patients were HBsAg-positive, and hepatitis C virus (HCV; Axsym,
Abbott Laboratories, IL, USA and Elecsys, Roche Diagnostics,
Mannheim, Germany), hepatitis D virus (HDV; Dia.Pro Diagnostic
Bioprobes, Milan, Italy), and HIV (Axsym, Abbott Laboratories, IL,
USA and Elecsys, Roche Diagnostics, Mannheim, Germany) markers
were negative. All of the patients had HBsAg positivity. None of the
patients had received the HBV vaccination prior to their diagnosis as
a chronic HBV carrier. Clinical and laboratory features of the study
patients according to the EASL clinical practice guidelines are listed
in Table 1.
8
mutations to ETV (rtI169T/sF161L and rtT184SL/sL176 V).
A
recent study has conferred new acronyms to these HBV pol/S
gene overlap mutants: ADAPVEMs, for antiviral drug-associated
potential vaccine-escape mutants, and ADASMs, for antiviral drug-
9
associated S gene mutants.
It has been demonstrated in a study on HBV-seronegative
chimpanzees that an in vitro generated polymerase gene mutant
containing a combination of three mutations (rtV173L, rtL180 M,
rtM204 V), two of which resulted in changes to the S gene (sE164D,
10
sI195 M), was infectious. To date, clinical evidence for the spread
11
or transmission of ADAPVEMs has been limited. No cases of
infection by ADAPVEMs have yet been reported in human
vaccinees, but the transmission of drug resistance-associated
HBV mutants to unvaccinated individuals has been reported;
however, the cases identified were infected by the LAM resistance-
associated rtM204 V/sI195 M mutant. Whether or not the infecting
12–14
HBV variants were naturally occurring is unknown.
Similarly,
ADAPVEMs (LAM-resistant mutations) have also been found in the
HBV isolated from Turkish hemodialysis patients with occult HBV
infection, and these patients were not undergoing antiviral
15
therapy.
2.2. DNA isolation and real-time PCR
The present study aimed to assess the prevalence and types of
ADAPVEMs in Turkish patients with CHB.
HBV DNA was detected and quantified from serum samples by a
commercial real-time PCR assay – COBAS Ampliprep/COBAS
TaqMan HBV test (Roche Diagnostics, Mannheim, Germany).
2
. Materials and methods
2.1. Patients
2.3. Sequencing of the HBV pol gene region
0
The study was carried out between March 2007 and July 2010 at
Briefly, a pair of primers was designed (forward: 5 -TCGTG
0
0
Kocaeli University Hospital and included patients already under
treatment during this time period and those who were started on
NUC therapy. The study involved all HBV DNA-positive samples
detected by PCR from a total of 442 patients. These patients were in
the following phases of HBV infection: immune tolerant (n = 50),
immune reactive (n = 37), inactive carrier (n = 90), HBeAg-negative
CHB (n = 217), and HBsAg-negative (n = 12), or were hemodialysis
patients (n = 36). One hundred eighty-six patients were receiving
nucleos(t)ide analogue (NUC) therapy and 256 patients had
treatment-na ı¨ ve CHB. The patients undergoing NUC therapy were
GTGGACTTCTCTCAATT-3
and
reverse:
5 -CGTTGACAGAC
0
TTTCCAATCAAT-3 ) for amplification of the HBV pol region. The
PCR conditions were: 95 8C for 10 min, and then 35 cycles
consisting of 95 8C for 45 s, 60 8C for 45 s, and 72 8C for 45 s. The
final concentration of the primers was 0.3
mM. The size of the
amplicon in HBV was around 742 bp, and included all the known
NUC resistance mutations in HBV. Phire Hot Start DNA polymerase
(Finnzymes Oy, Vantaa, Finland) was used in the sequencing
protocol. All PCR products were purified using the High Pure PCR
Product Purification Kit (Roche Diagnostics, Mannheim, Germany).
Sequencing was carried out using an ABI PRISM 3130 Genetic
Analyzer (Applied Biosystems Inc., Foster City, CA, USA). The
BigDye Terminator v3.1 Cycle Sequencing Kit (Amersham Phar-
macia Biotech Inc., Piscataway, NJ, USA), 36 cm capillary array, and
1
being treated with oral LAM (Zeffix 100 mg/day; GlaxoSmithKline,
1
Middlesex, UK), ADV (Hepsera 10 mg/day; Gilead Sciences, Inc.,
1
Foster City, CA, USA), ETV (Baraclude 0.5 mg/day and 1 mg/day;
1
Bristol-Myers Squibb Co., Princeton, NJ, USA), and TDF (Viread
Table 1
Clinical and laboratory characteristics of the patients by study group
a
Characteristic
Immune tolerant
phase
Immune
Inactive carrier
HBeAg-negative CHB
HBsAg-negative
phase
Hemodialysis
reactive phase
No. of patients
50
37
90
217
12
36
Sex (M/F)
32/18
21/16
47/43
142/75
9/3
24/12
Age, median (range) years
ALT, median (range) U/l
HBV DNA, median (range) IU/ml
45 (10–64)
59.5 (18–1082)
33.5 (4–58)
40.5 (14–157)
27 (2–78)
17.5 (10–59)
29.5 (4–81)
49 (12–537)
44 (28–76)
25 (16–498)
35.5 (17–80)
7.5 (1–77)
6
4
2
6
3
3
>1.16 ꢁ 10
1.34 ꢁ 10
1.15 ꢁ 10
5.00 ꢁ 10
5.10 ꢁ 10
4.4 ꢁ 10
6
8
2
5
1
3
3
8)
2
4
1
4
(
10 –>10 )
(10 –10 )
D (100%)
(10 –10 )
D (100%)
(10 –>10
(10 –10 )
(10 –10 )
D (100%)
HBV genotype (%)
D (100%)
D (99.5%) A (0.5%)
D1, 183 (84%)
D2, 24 (11%)
D3, 8 (4%)
D (100%)
HBV subgenotype, n (%)
D1, 47 (94%)
D2, 1 (2%)
D3, 2 (4%)
D1, 31 (84%)
D2, 4 (11%)
D3, 2 (5%)
D1, 73 (81%)
D2, 9 (10%)
D3, 8 (9%)
D1, 12 (100%)
D1, 25 (70%)
D2, 8 (22%)
D3, 3 (8%)
D4, 1 (0.5%)
A2, 1 (0.5%)
HBeAg, hepatitis B e antigen; CHB, chronic hepatitis B; HBsAg, hepatitis B surface antigen; M, male; F, female; ALT, alanine aminotransferase.
a
No HBeAg data were obtained from the hemodialysis study group; however the median duration of dialysis in these patients was 10.5 years (range 5 months–27 years).

e724
M. Sayan, S.C. Akhan / International Journal of Infectious Diseases 15 (2011) e722–e726
TM
Table 2
POP-7 polymer (Applied Biosystems Inc.) were used. Sequencing
was carried out according to the manufacturer’s protocol (http://
www3.appliedbiosystems.com).
Laboratory data and clinical features of ADAPVEM group patients
Characteristic
ADAPVEM
No. of patients
46
2.4. Determination of HBV genotype/subgenotype and pol/S gene
Sex (M/F)
30/16
mutations
Age, median (range) years
ALT, median (range) U/l
HBV DNA, median (range) IU/ml
HBV genotype (%)
50.5 (24–77)
31.5 (13–537)
5
2
8
5.02 ꢁ 10 (10 –>10 )
The Genafor/Arevir–geno2pheno drug resistance tool (Center of
D (100%)
Advanced European Studies and Research, Bonn, Germany; http://
coreceptor.bioinf.mpi-inf.mpg.de/) for HBV is a database that is
specifically designed for rapid computer-assisted virtual pheno-
typing of HBV, and accepts genome (nucleic acid) sequences as
input. Geno2pheno searches for homology between input
sequences and others already stored in its database, which also
stores relevant clinical data for HBV genotype/subgenotype and
drug resistance/S gene mutations. The data accumulated by direct
sequencing were analyzed either manually or using the geno2-
pheno tool for drug resistance and S gene mutations. The
overlapping pol/S gene segments were searched for 344/266
amino acid codons in the reverse transcriptase (rt) domain of the
pol gene.
HBV subgenotype, n (%)
D1; 38 (83%)
D2; 3 (6%)
D3; 4 (9%)
D4; 1 (2%)
Clinical status
Immune tolerant phase, n = 8 patients
Immune reactive phase, n = 3 patients
Inactive carrier, n = 6 patients
HBeAg-negative CHB, n = 28 patients
HBsAg-negative phase, n = 1 patient
Treatment-na ı¨ ve, n = 2 patients
Undergoing treatment, n = 44 patients
Treatment status
ADAPVEM, antiviral drug-associated potential vaccine-escape mutant; M, male; F,
female; ALT, alanine aminotransferase; HBeAg, hepatitis B e antigen; CHB, chronic
hepatitis B; HBsAg, hepatitis B surface antigen.
2
.5. Statistical analysis
(rtL180 M + rtS202C + rtM204 V + rtV214P/sI195 M). The preva-
lence of ADAPVEMs was 24% (44/186) in those undergoing NUC
therapy and 0.7% (2/256) in the treatment-na ı¨ ve group; this
difference was significant (Pearson Chi-square, p = 0.00).
Age, HBV DNA load, and alanine aminotransferase (ALT) levels
were treated as numeric variables; gender, clinical phase of CHB,
and ADAPVEM were treated as categorical variables. The signifi-
cance of the difference between two numeric variables was
compared using a Levene’s test for equality of variances. The
significance of the difference between two proportions was
measured using the Pearson Chi-square test or Fisher’s exact test.
p-Values of ꢂ0.05 were considered statistically significant.
Statistical analyses were done using SPSS v.13.0.0 for Windows
rtA181T/sW172* was found in 2% (1/46) of patients in whom
ADAPVEMs were detected and in 11% (1/9) of acyclic phosphonate
(ADV)-resistant cases (Table 3). However, none of the patients with
HCC related to HBV had rtA181T/sW172* mutation combination.
The treatment-na ¨ı ve patients had a median HBV viral load of
4
1
8
5
1
5.22 ꢁ 10 (range 10 –>10 ) compared to 5.75 ꢁ 10 (range 10 –
8
>10 ) in patients undergoing treatment; treatment-na ı¨ ve patients
(SPSS Inc., Chicago, IL, USA).
had a median ALT value of 17 (1–515) U/l compared to 180 (12–
1082) U/l in patients undergoing treatment. According to Levene’s
3
. Results
test for equality of variances, the difference in the median HBV
viral load value and ALT levels between treatment-na ¨ı ve patients
and those undergoing treatment were significant (p = 0.001 and
p = 0.012). However, these values were not found to be closely
related to ADAPVEMs. In addition, median age and gender ratios
(male/female) were 55 (2–80) years and 39 (4–81) years, and 154/
102 and 121/65, in treatment-na ¨ı ve patients and patients
undergoing treatment, respectively. The difference in median
age was significant, but the difference in gender variation was not
significant between the two groups (Levene’s test for equality of
All of the ADAPVEMs detected in patients undergoing treatment
occurred in those with background LAM therapy (LAM mono-
therapy or ADV in addition to LAM therapy). Two of these patients
had HCC related to HBV and three patients had cirrhosis (one
patient had compensated cirrhosis and two had decompensated
cirrhosis). Eighty of the patients who received LAM therapy had
experience with peginterferon alpha-2b therapy. Three of these
patients developed virologic relapse at the end of 48 weeks of
treatment, four patients developed virologic resistance during
treatment, and one of the patients had no virologic response at the
end of 24 weeks of treatment. Clinical and laboratory character-
istics of the ADAPVEM group of patients are shown in Table 2.
Seven types of ADAPVEM were detected in the total CHB
patients: rtM204 V/sI195 M, rtM204I/sW196S, rtM204I/sW196L,
Table 3
Emergence of ADAPVEM according to the nucleos(t)ide analogues used to treat the
patients
a
Nucleos(t)ide analogue
L-nucleosides
pol gene mutation S gene mutation n (%)
rtV173L
rtM204V
rtM204I
rtA181T
rtA181T
rtA181V
rtV191I
sE164D
5 (11)
11 (24)
25 (54)
1 (2)
2 (4)
6 (13)
ND
rtV173L/sE164D,
rtA181T/sW172*,
rtA181T/sW172L,
and
(LAM, LdT)
sI195M
rtA181 V/sL173F (Table 3). The ADAPVEMs were associated with
LAM, LdT, and ADV. The prevalence of ADAPVEMs in all CHB
patients was found to be 10% (46/442). With regard to CHB clinical
phase, ADAPVEM prevalence was found to be 16% (8/50) in the
immune tolerant phase, 8% (3/37) in the immune reactive phase,
sW196S/L
sW172*
Acyclic phosphonates
(ADV, TDF)
sW172L
sL173F
sW182*
Deoxyguanosine analogues rtI169T
sF161H/L
sL175F + sL176V
sL175F
ND
7
8
% (6/90) in inactive carriers, 13% (28/217) in HBeAg-negative CHB,
% (1/12) in HBsAg-negative phase, and 0% (0/36) in hemodialysis
(ETV)
rtT184C
ND
rtT184L/S
rtT184G/M
rtS202C/G
rtS202I
ND
sL176V/*
sS193F/L
sS194F/S
ND
patients. The difference in prevalence of ADAPVEMs across the
different CHB clinical phases was not significant (Pearson Chi-
square, p = 0.112).
ND
ND
ADAPVEM, antiviral drug-associated potential vaccine-escape mutant; LAM,
lamivudine; LdT, telbivudine; ADV, adefovir; TDF, tenofovir; ETV, entecavir; ND,
not detected.
Two of the patients detected with ADAPVEMs were treatment-
na ¨ı ve: patient 1 had primary LAM, LdT, and intermediate ETV
drug resistance (rtL80I + rtM204I + rtQ215S/sW196L) and patient
a
Some patients had multiple mutations, however the percentage of the mutation
2
had primary LAM, LdT, and ETV drug resistance
was calculated for 46 patients.

M. Sayan, S.C. Akhan / International Journal of Infectious Diseases 15 (2011) e722–e726
e725
variances: p = 0.014 and p = 0.091, respectively). However, neither
age nor gender was correlated with the presence of detected
ADAPVEMs.
Direct sequencing results revealed HBV of genotype D in all
patients, except one patient with genotype A (A2, 0.2% (1/442))
who was an HBeAg-negative CHB patient in the treatment-na ¨ı ve
group. According to the results of the HBV subgenotyping analysis,
D1 was predominant, found in 84% (371/442) of all study patients,
and subgenotypes D2, D3, and D4 were found in 10% (46/442), 5%
In Turkey, about 6500 individuals per year are newly infected
20
with HBV, showing intermediate (2–7%) endemicity. However,
little is known about HBsAg escape mutations. Recently, the first
HBV vaccine escape mutation (sT143*) was identified in a child
2
1
with CHB in Turkey. A study with sequencing of the amplified
surface gene region has suggested sM125T and sT127P mutations
for HBsAg escape in Turkish patients with CHB and their family
2
0
members. A recently published study reported a diagnostic
escape HBsAg mutation (sS143L) causing chronic HBV infection in
22
(
23/442), and 0.2% (1/442), respectively (Table 1).
. Discussion
In chronically infected individual, the extent of HBV
a previously vaccinated treatment-na ı¨ ve Turkish patient. How-
ever, recently we have seen the monitoring of the prevalence and
pattern of the typical mutations for HBsAg escape and concomitant
drug resistance mutations in patients undergoing NUC therapy and
4
2
3
a
in treatment-na ı¨ ve Turkish patients with CHB. In this study,
ADAPVEMs in Turkish patients with CHB were discussed for the
12
16
replication is considerable, reaching >10 virions per day. As
pol is an rt that lacks proof-reading capacity, HBV replication is also
associated with a high mutational rate of 10 substitutions/base/
cycle. Thus, all possible single-base changes in the HBV genome
are generated daily, thereby accounting for the observation that
mutations associated with NUC resistance and/or HBsAg escape
first time. However, because of high perinatal transmission of HBV
ꢀ5
24
infection,
ADAPVEMs should be monitored in southeastern
1
7
Turkey among treated women of childbearing age, especially in
those undergoing LAM therapy. Neither HBIg nor active immuni-
zation would prevent infection with ADAPVEM transmitted from
13
9
exist in patients prior to therapy.
mother to child.
Successful therapy should be aimed at suppressing all existing
viral variants, thus preventing the selection of minority species and
In conclusion, we have revealed ADAPVEMs across the different
phases of CHB in Turkish patients. Preferred drugs in Turkey, such
as lamivudine, have the potential to cause the emergence of
ADAPVEMs, with the possibility that these will spread to both
individuals immunized with the hepatitis B vaccine and non-
immunized individuals. ADAPVEMs should be monitored in
infected and treated patients and their public health risks assessed.
Conflict of interest: This study was not funded by any
organization. No conflict of interest to declare.
18
their subsequent evolution. The genomic changes associated
with drug resistance in HBV variants can be stable and these
9
,12
resistant viruses can be transmitted to another individual.
In
populations where LAM has been used widely to treat patients
continuously for periods of several years, as in Turkey (LAM is
relatively inexpensive and an obligatory drug at the start of
treatment in Turkey), viruses with alterations in the S gene are
likely to occur relatively frequently, and some will be ADAPVEMs.
The current results reveal ADAPVEMs associated with LAM
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ADAPVEMs associated with LAM and LdT were found to be
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rtM204I/V + sI195 M/sW196S/L mutations in LAM-resistant cases
in the SeqHepB database have also been shown to be predomi-
1
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However, ADAPVEMs associated with ADV (rtA181T/
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3). One of the drug-resistant mutants, rtA181T/sW172*, has been
7
shown to have oncogenic potential in transgenic mice. The
rtA181T/sW172* variant was detected in the SeqHepB database in
1
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1
% and 11% of LAM- and ADV-resistant cases, respectively. We
also detected the rtA181T/sW172* variant at nearly the same rate:
% and 11% of LAM- (predominant in ADAPVEM detected cases)
and ADV-resistant cases, respectively.
1
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