Molecular Characterization of Ambiguous Mutations in HIV-1 Polymerase Gene: Implications for Monitoring HIV Infection Status and Drug Resistance
Detection of recent HIV infections is a prerequisite for reliable estimations of transmitted HIV drug resistance (t-HIVDR) and incidence. However, accurately identifying recent HIV infection is challenging due partially to the limitations of current serological tests. Ambiguous nucleotides are newly emerged mutations in quasispecies, and accumulate by time of viral infection. We utilized ambiguous mutations to establish a measurement for detecting recent HIV infection and monitoring early HIVDR development. Ambiguous nucleotides were extracted from HIV-1 pol-gene sequences in the datasets of recent (HIVDR threshold surveys [HIVDR-TS] in 7 countries; n=416) and established infections (1 HIVDR monitoring survey at baseline; n=271). An ambiguous mutation index of 2.04×10(-3) nts/site was detected in HIV-1 recent infections which is equivalent to the HIV-1 substitution rate (2×10(-3) nts/site/year) reported before. However, significantly higher index (14.41×10(-3) nts/site) was revealed with established infections. Using this substitution rate, 75.2% subjects in HIVDR-TS with the exception of the Vietnam dataset and 3.3% those in HIVDR-baseline were classified as recent infection within one year. We also calculated mutation scores at amino acid level at HIVDR sites based on ambiguous or fitted mutations. The overall mutation scores caused by ambiguous mutations increased (0.54×10(-2)3.48×10(-2)/DR-site) whereas those caused by fitted mutations remained stable (7.50-7.89×10(-2)/DR-site) in both recent and established infections, indicating that t-HIVDR exists in drug-naïve populations regardless of infection status in which new HIVDR continues to emerge. Our findings suggest that characterization of ambiguous mutations in HIV may serve as an additional tool to differentiate recent from established infections and to monitor HIVDR emergence.
Molecular Characterization of Ambiguous Mutations in
HIV-1 Polymerase Gene: Implications for Monitoring HIV
Infection Status and Drug Resistance
Du-Ping Zheng1, Margarida Rodrigues2, Ebi Bile3, Duc B. Nguyen4, Karidia Diallo1, Joshua R. DeVos1,
John N. Nkengasong1, Chunfu Yang1*
1 Division of Global HIV/AIDS, Center for Global Health, Centers for Disease Control and Prevention (CDC), Atlanta, Georgia, United States of America, 2 CDC-
GAP Angola, Luanda, Angola, 3 CDC-GAP Botswana, Gaborone, Botswana, 4 Department of Health and Human Services/US CDC, Hanoi, Vietnam
Detection of recent HIV infections is a prerequisite for reliable estimations of transmitted HIV drug resistance (t-
HIVDR) and incidence. However, accurately identifying recent HIV infection is challenging due partially to the
limitations of current serological tests. Ambiguous nucleotides are newly emerged mutations in quasispecies, and
accumulate by time of viral infection. We utilized ambiguous mutations to establish a measurement for detecting
recent HIV infection and monitoring early HIVDR development. Ambiguous nucleotides were extracted from HIV-1
pol-gene sequences in the datasets of recent (HIVDR threshold surveys [HIVDR-TS] in 7 countries; n=416) and
established infections (1 HIVDR monitoring survey at baseline; n=271). An ambiguous mutation index of 2.04×10-3
nts/site was detected in HIV-1 recent infections which is equivalent to the HIV-1 substitution rate (2×10-3 nts/site/year)
reported before. However, significantly higher index (14.41×10-3 nts/site) was revealed with established infections.
Using this substitution rate, 75.2% subjects in HIVDR-TS with the exception of the Vietnam dataset and 3.3% those
in HIVDR-baseline were classified as recent infection within one year. We also calculated mutation scores at amino
acid level at HIVDR sites based on ambiguous or fitted mutations. The overall mutation scores caused by ambiguous
mutations increased (0.54×10-23.48×10-2/DR-site) whereas those caused by fitted mutations remained stable
(7.50-7.89×10-2/DR-site) in both recent and established infections, indicating that t-HIVDR exists in drug-naïve
populations regardless of infection status in which new HIVDR continues to emerge. Our findings suggest that
characterization of ambiguous mutations in HIV may serve as an additional tool to differentiate recent from
established infections and to monitor HIVDR emergence.
Citation: Zheng D-P, Rodrigues M, Bile E, Nguyen DB, Diallo K, et al. (2013) Molecular Characterization of Ambiguous Mutations in HIV-1 Polymerase
Gene: Implications for Monitoring HIV Infection Status and Drug Resistance. PLoS ONE 8(10): e77649. doi:10.1371/journal.pone.0077649
Received April 23, 2013; Accepted September 12, 2013; Published October 17, 2013
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by
anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Funding: This research has been supported by the President’s Emergency Plan for AIDS Relief (PEPFAR) through the Centers for Disease Control and
Prevention. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
* E-mail: CYang1@cdc.gov
With over 8.2 million HIV-infected patients on antiretroviral
therapy (ART) in low- and middle-income countries at the end
of 2011, emergence and transmission of HIV drug resistance
(HIVDR) are ongoing public health challenges in the battle
against HIV/AIDS [1-6]. Because of the incomplete suppression
of viral replication by ART [2-4,7], even with the optimal
adherence, HIVDR may still emerge in ART-patients; whereas
under suboptimal ART situations, such as incomplete ART
compliance and/or improper practice of regimen prescriptions,
development of HIVDR could be enhanced . Currently,
mutations associated with HIVDR at 85 sites are identified,
including 32 in reverse transcriptase (RT, 16 for nucleoside RT
inhibitors [NRTIs] and 16 for non-nucleosides RTI [NNRTIs]),
36 in protease, seven in envelope, and 10 in integrase genes
Estimates of transmitted HIVDR (t-HIVDR) and HIV
incidence have been two important surrogates in measuring
ART efficacy and prevention program effectiveness . To
assess the t-HIVDR in resource-limited countries, the World
Health Organization (WHO) recommends conducting HIVDR
threshold survey (HIVDR-TS) in recently HIV-infected
populations enrolled by using WHO criteria [11,12]. Likewise,
estimate of HIV incidence also requires recent infections that
are determined by using serological assays with cross-
sectional HIV-positive samples at population level [13-15].
Nevertheless, studies have indicated that these assays
overestimated HIV incidences due to false classification of
recent infections in certain populations and lack of validated
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standards in accurately distinguishing recent (within 1 year)
from established infections [13,14,16].
HIV evolves rapidly attributed partially to the high error rate
of its RT [17,18], and accumulates mutations at a certain rate
over time . This results in generating a large number of
variants (quasispecies) in a host and increasing genetic
diversity in a viral population . By analyzing the genetic
relationships among quasispecies, the dynamic evolutional
pathway of a variant can be tracked on a time scale within and
among hosts [21,22]. In the early stage of viral replication, HIV
variants with new point mutations account for only a small
proportion of the total wild-type populations, thus a particular
point mutation at an allele is detected as a mixture along with
the wild-type by conventional population-based (Sanger)
sequencing, which is termed as ambiguous mutation/
nucleotide. By applying the defined nucleotide substitution rate
of HIV-1 [19,20], the quantified ambiguous mutations within a
sequence could be used to estimate the duration of an HIV
infection. Recent studies have demonstrated a constant
increase of ambiguous nucleotides during the first 8 years of
HIV infections at a rate of 0.2% per year in HIV subtype B pol-
gene , and 0.45-0.5% of ambiguous nucleotides as a cutoff
for distinguishing recent from established infections [23-25]. In
this study, we characterized ambiguous nucleotides in non-B
subtype sequences from eight population-based HIVDR-TS
and HIVDR monitoring surveys and determined a predictive
value for estimating HIV infection status using a molecular
evolutional approach and compared ambiguous mutation
evolving rate between sequences generated from non-B (A, C,
and CRF01_AE) and B subtype viral strains, and evaluated the
accuracy of WHO epidemiological criteria for the recent
infection determination. We also used this approach to monitor
HIVDR development at the early stage of HIV infections.
Materials and Methods
Data sources and types
Sequence data were from HIVDR-TS conducted in seven
countries (Angola, Botswana, China, Kenya, Malawi, Tanzania,
and Vietnam) (n=416) and from a baseline survey (n=271) in
monitoring HIVDR development in patients commencing ART
in Nigeria during 2005-2009 (Table 1). The detail demographic
and clinical data of participants were previously described
[26-32]. In brief, all patients in HIVDR-TS were enrolled as
recent infections according to WHO criteria [11,12]. For those
from Angola, Botswana, Kenya, Malawi and Tanzania, they
were pregnant women who were <25 years old, attending
antenatal clinics (ANC), diagnosed with HIV infections for the
first time, and ART-naïve; and for those from China and
Vietnam, they were individuals attending voluntary counseling
and testing (VCT) sites and were partially intravenous drug
users (IDU). The WHO criteria were designed to increase the
likelihood of identifying recently infected individuals for the
HIVDR-TS. The participants in baseline survey were patients
eligible for ART according to the Nigeria national guidelines for
HIV/AIDS care and treatment in adolescents and adults (CD4
≤200 cells/µl, WHO stage III or IV or AIDS defined illness) ,
they were most likely established or chronic HIV-infected
individuals. This dataset was used to compare to those of
recent infections in the HIVDR-TS for ambiguous mutation
We also included a dataset of subtype B sequences (n=63)
from published resources  for the comparison of ambiguous
mutation preference with our non-B subtype sequences. These
sequences were generated from individuals who had been
infected within 155 days confirmed by serological tests.
DNA sequencing, genotyping and subtyping
All partial pol-gene sequences were generated using a
validated HIV-1 genotyping assay using a conventional
population-based bi-directional sequencing procedure [34-36].
The lengths of sequences were 981 (HIVDR-TS data) and
Table 1. Characteristics of sequence datasets and subtyping in partial HIV-1 pol gene.
CountryaYear Data source Infection route Sequences Subtypeb
A B C G CRF01_AE Others Untypeable
Angola 2009 ANC (HIVDR-TS) heterosexual 39 9 5 5 20
Botswana 2007 ANC (HIVDR-TS) heterosexual 134 132* 2
China 2006 VCT (HIVDR-TS) Multi-routes 45 8 3 20* 9 5
Kenya 2005-2006 ANC (HIVDR-TS) heterosexual 33 19* 2 1 11
Malawi 2006 ANC (HIVDR-TS) heterosexual 52 51* 1
Tanzania 2005-2006 ANC (HIVDR-TS) heterosexual 45 14* 17 4 10
Vietnam 2006-2008 VCT (HIVDR-TS) Multi-routes 68 68
Nigeria 2008 ET (T1 baseline) unknown 271 5 1 135
Canada 2002-2008 Early infection unknown 63 63*
a Country data used in this study were collected from HIV drug resistance threshold survey (HIVDR-TS), T1 baseline survey, and published data (24) respectively; Antenatal
care (ANC), Voluntary counseling and testing (VCT), Eligible for treatment (ET); Numbers labeled with asterisk (*) were sequences selected to represent subtypes for
statistical analysis, see Table 3; b HIV-1 subtypes were primarily determined using REGA HIV subtyping tool (http://www.bioafrica.net/rega-genotype/html/subtypinghiv.html).
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1,002 (Baseline HIVDR monitoring survey data) nucleotides
(nts) containing HIVDR mutation sites of protease and RT
Sequences were primarily subtyped using Stanford REGA
HIV-1 Subtyping Tool version 2.0 (http://
dbpartners.stanford.edu/RegaSubtyping/). The sequences
used in this study included those published previously
(JQ617150-JQ617250), and new submissions (JX083986-
Detection and analysis of ambiguous mutations
Ambiguous mutations, which consist of mixed nucleotides at
a sequence position and named using the standard IUPAC
ambiguous nucleotide codes, were determined and
automatically called using customized software, Recall 
when the sequencing signal intensity of the minor base was
≥20% of the major base signal at a nucleotide position on bi-
directional sequences after subtracting background noise.
Ambiguous mutations were extracted from each of sequences
and tallied at country level. The mean of the ambiguous
mutations was then calculated using the formula: MAM = ∑
NAM/N (MAM: mean of ambiguous mutations per sequence; NAM:
number of ambiguous mutations of a sequence; ∑NAM: sum of
ambiguous mutations in a dataset, N: total number of
sequences in the dataset). The index (I) of ambiguous
mutations was calculated using the formulas: IAM = NAM/Ls for
an index at sequence level, or IAM = MAM/Ls for an index at a
dataset level (IAM: index of ambiguous mutations per site; Ls:
length of a sequence by nucleotide), (note: Ls is 1/3 of full
length when calculation was for 1st, 2nd, or 3rd codon
The composition of nucleotides or ambiguous nucleotides in
a sequence dataset was obtained using BioEdit with the
algorithm of base composition and mass export .
Ambiguous mutations were then stratified at 1st, 2nd, 3rd and
all codon positions by dataset of threshold, baseline and
Vietnam (VT), or by HIVDR and non-HIVDR sites based on the
2013 HIVDR List . The index of ambiguous mutation was
calculated using the same formulas as described previously.
At the AA level, we scored 1 for a pure mutated AA and 0.5
for an ambiguous mutated AA because of its ambiguity, and
calculated the total DR mutation score at each of the HIVDR
sites  with the formula: DR mutation % = ([NMAA+NAMAA/2]/
NSEQ)×100% (NMAA: number of mutated AA; NAMAA: number of
ambiguous mutated AA).
Recent infection determination
Based on the estimated HIV nucleotide substitution rate of
2×10-3 nts per site per year [19,20], a sequence with ≤2
ambiguous mutations, or 2×10-3 ambiguous nucleotides per
site, was considered to be derived from a subject who was
infected within one year. In a dataset, the percentage of recent
infections was assessed by calculating the proportion of
sequences that had ≤2 ambiguous mutations.
Statistical analyses were performed using IBM SPSS
Statistics 20 (IBM), or otherwise were indicted. Data of non-
normal distribution were determined by one-Sample
Kolmogorov-Smirnov Test. Plot of dataset median, interquartile
range (IQR), and range with and without outliers was made
using online resource (http://www.physics.csbsju.edu/stats/).
The overall significant difference of values in all datasets was
determined by Kruskal-Wallis test, and the difference of
pairwise comparison was determined by Mann-Whitney test
when Kruskal-Wallis P value was <0.05. For multiple
comparisons, the P values were corrected by the Bonferroni
This is a data mining study based on the sequences
generated from our previously published survey studies [26-32]
in which all the surveys had been approved by the local
Institutional Review Board (IRB) from Angola, Botswana,
China, Kenya, Malawi, Tanzania, Vietnam, and Nigeria as well
as the Associate Director for Science at the Center for Global
Health of CDC, USA who determined that the anonymous
specimen testing performed at CDC was a non-human subject
Range and index of ambiguous mutations for country-
Ambiguous mutations were extracted from 8 datasets of a
total of 687 sequences (Table 1) and were used for statistical
descriptive and significant analyses (Figure 1). We observed
that two HIVDR-TS datasets (Angola and China) each
contained one sequence with much higher number of
ambiguous mutations, 25 and 19, respectively than those in the
remaining HIVDR-TS sequence datasets (Figure 1, indicated
by dash box). For study purpose, we analyzed the two datasets
with and without the outlier sequences.
For datasets from Angola, Botswana, China, Kenya, Malawi
and Tanzania, the maximal range of ambiguous mutations
without the two outliers was 0-15 nts with mean ranging from
1.13-2.68 nts per sequence. However, for those from Vietnam
and Nigeria, the ranges were 0-38, and 0-82 nts with means of
17.96 and 14.42 nts per sequence, respectively. Likewise, the
ranges of ambiguous mutation index were 1.16-2.74×10-3 nts
per site among the datasets of Angola, Botswana, China,
Kenya, Malawi and Tanzania; whereas the ranges of
ambiguous mutation index for datasets from Vietnam and
Nigeria were 18.32 and 14.40×10-3 nts per site, respectively
When the two outliers were included in the analysis, the
mean and index of ambiguous mutations were 3.26 nts per
sequence and 3.31×10-3 nts per site for Angola, and 2.91 nts
per sequence and 2.96×10-3 nts per site for China, respectively,
which were slightly higher than the values generated without
outlier sequences but they were still much lower than those
from Vietnam and Nigeria (3.26 and 2.91 vs 17.96 and 14.42
for the mean of ambiguous mutations and 3.31 and 2.96 vs
18.32 and 14.40×10-3 nts per site for the index of ambiguous
mutations, Figure 1).
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Statistical analyses indicated that both mean and index of
ambiguous mutation in the HIVDR-TS datasets (likely from
recent infections) were significantly lower than those in the
HIVDR monitoring baseline survey (established infection)
(p<0.001) with an exception of those from Vietnam which is
described in the later sections.
Ambiguous mutation in recent and established HIV
To calibrate the difference of ambiguous mutations between
recent and established HIV infections on a larger scale, we
reorganized the data into three subsets: 1) threshold surveys
for which samples were collected mainly from heterosexually
transmitted individuals (N= 346), 2) baseline (N=271), and 3)
Vietnam (N=68), and characterized the ambiguous mutations at
each setting (Table 2). For the subset of threshold surveys, the
mean of ambiguous mutations was 2.00 nts with a range of
0-15 nts per sequence, and the index was 2.04×10-3 (95%
confidence intervals, [CI]: 1.13-2.71×10-3) ambiguous mutations
per site. However, the subset from Vietnam, which was also
from threshold surveys but might consist of subjects with
different routes of transmission [30,39], and the one from
Nigeria, which contained established and chronic infections,
yielded means of 17.96 and 14.42 ambiguous mutations per
sequence, and index of 18.30 (95% CI: 16.59-20.02) and 14.40
(95% CI: 13.50-15.29) ×10-3 ambiguous mutations per site
(p=0.538), respectively, which were significantly higher than the
values of the threshold data (p<0.001) (Table 2, Figure 2D).
These combined data further confirmed that ambiguous
Figure 1. Descriptive statistics of ambiguous mutation in various sequence datasets. Plot of ambiguous mutations with
descriptive statistics was performed using online statistical tool (http://www.physics.csbsju.edu/stats/). Individual country dataset
was described for minimal and maximal ranges (short horizontal line at the bottom and top of the box), interquartile range (IQR, at
1st to 3rd quartile, box), median (line inside box), suspected outlier (open dot), and outlier (solid dot). Number in the bracket is the
number of sequences from the country, Angola (AO), Botswana (BW), China (CN), Kenya (KE), Malawi (MW), Tanzania (TZ),
Vietnam (VN), Nigeria (NG), and Canada (CA) . Numbers with asterisk were calculated without the outlier in dash square box.
Figure 1-insert shows the descriptive statistics of ambiguous mutation index in the dataset based on subtype (Table 3).
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mutation index was significantly lower in the individuals in the
HIVDR-TS than those in the baseline and Vietnam surveys.
We further characterized the sequence distribution by
ambiguous mutations. Results exhibited completed different
patterns between the 3 datasets (Figure 2A-C). In the threshold
survey subset, the highest distribution of sequences peaked at
0 ambiguous mutation and 80% of the sequences had <3
ambiguous mutations; however, the peak of distribution curve
shifted to the range of 3-28 ambiguous mutations in the
baseline subset, and diversified with various cluster ranges of
ambiguous mutation in the Vietnam subset (Figure 2A-C).
These patterns of distribution curve indicated the uniformity of
infection status among the subjects in the dataset: the narrower
the distribution range is, the more uniformity the subjects are
and a wide and diversified distribution pattern suggests a wide
range of mixed infections, e.g. those shown in VN dataset. By
using the estimated HIV nucleotide substitution of 2×10-3 nts
per site per year [19,20], sequences of 75.2% in threshold,
3.3% in baseline, and 10.3% in Vietnam subsets could be
classified as coming from people infected with HIV within one
year. This result implies that around 75% of patients enrolled in
the HIVDR-TS using WHO epidemiological criteria could be
recently infected individuals.
Ambiguous mutation at HIV drug resistant and non-
To explore if there was any site preference for ambiguous
mutation to occur, we measure the ambiguous mutation at DR
or non-DR sites and all codon positions  . In general, no
significant difference of ambiguous mutation was observed
between DR and non-DR sites of all codon positions within
each of the subsets (p=0.889 [HIVDR-TS], 0.590 [baseline],
and 0.110 [VN-TS]) (Table 2), indicating a random mutation
mechanism. However, at the 1st codon position the ambiguous
mutation at DR sites was always higher than the non-DR sites
across the datasets which was in reverse at the 2nd codon
position. For example, in the threshold subset, the ambiguous
mutation at the DR sites was 56.7% higher at the 1st codon
position but 45.5% lower at the 2nd codon position than those
at the non-DR sites. At the 3rd codon position, the ambiguous
mutation between the DR and non-DR sites varied. They were
similar in threshold (4.24 and 4.16), but were higher at the non-
DR sites than the DR sites for the baseline and Vietnam
subsets (33.05 vs 25.71; 42.50 vs 32.21, respectively),
implying the evolutional pressure applied to DR and non-DR
codon positions is different during the course of viral replication
Amino acid fitness of ambiguous and mutated
nucleotides at HIV DR sites
To obtain the DR-score attributed to the ambiguous or non-
ambiguous mutated nucleotides, we stratified the non-
synonymous DR-associated mutations at amino acid (AA) level
based on the 2013 HIVDR list  (Figure 3). Results showed
that 12 of the 65 DR sites had patterns on the mutation score.
It was constantly high at 3 DR sites (M36, 76.36-99.07%; H69,
90.10-98.53%; L89, 71.61-98.34%), moderate at 1 DR site
(L63, 40.17-56.62%), and low at 1 DR site (V179,
10.52-20.59%) across all 3 subsets. The mutation score
increased substantially at 2 DR sites (V82, 0.432.2148.52%;
Q151, 0.2998.5398.52%), and decreased at 5 DR sites (K20,
20.3919.854.06%; D60, 15.375.882.58%; T74,
Table 2. Characteristics of ambiguous mutations (AMs) and rates between threshold and baseline sequence datasets.
Threshold Baseline Vietnam-TS p-value
Number of Sequences 346 271 117
Sequence length (nts) 981 1002 867
AMs and occurrence rate
AM Range 0-15 0-82 0-38
Mean (AMs/sequence) 2.00 14.42 12.78
Rate (AMs/site, ×10-3) (95% C.I.) 2.04 (1.13-2.71) 14.40 (13.50-15.29) 14.74 (13.38-16.09) <0.001 (TS vs BL, VN); 0.538 (VN vs BL)
AM occurrence rate (AMs/site, ×10-3)
All codon positions DR vs nDR: 0.889 (TS), 0.590 (BL), 0.441 (VN)
Non-DR sites (nDR) 2.02 14.97 14.35
DR sites 2.07 12.09 12.26
1st codon position
Non-DR sites 0.90 6.76 5.86
DR sites 1.41 7.21 8.97
2nd codon position
Non-DR sites 1.01 5.06 5.34
DR sites 0.55 3.34 3.39
3rd codon position
Non-DR sites 4.16 33.05 31.86
DR sites 4.24 25.71 24.42
a The AMs of 2 outlier sequences were excluded for calculation.
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12.790.743.32%; V77, 15.370.742.40%; I93,
62.1522.791.85%) in the order of threshold, Vietnam and
baseline subsets. Interestingly, of these 12 DR sites, 10 were
located in the protease gene and only two (Q151 and V179)
were in the RT gene. Some might associate with polymorphism
or have combination effects on DR and viral replication
depending on subtypes [40,41]. It was also evident that the DR
score of ambiguous mutated AAs were gradually accumulating
in the Vietnam and baseline sequences comparing to the
By calculating the overall DR-associated mutations derived
from pure mutated AAs or ambiguous mutated AAs (Figure 4),
we found that the index of pure mutated AAs was constant
across the 3 subsets (7.50-7.89×10-2 per DR site) (p=0.681); in
contrast, the index of ambiguous mutated AAs increased
significantly from 0.54×10-2 per DR site for threshold to
4.30-3.48×10-2 per DR site for Vietnam and baseline datasets
(p<0.001), indicating that background of t-HIVDR existed in
ART-naïve populations of these cohorts. Under such
background, new HIVDR continued to develop in the studied
Subtyping and ambiguous mutation preference
Subtype of HIV sequences in each dataset was primarily
determined using the online REGA HIV subtyping tool (Table
1). A single dominant subtype was found in Botswana and
Malawi (subtype C), Kenya (subtype A), Nigeria (subtype G),
China and Vietnam (CRF01-AE); whereas multiple subtypes
and recombinants were identified in Tanzania and Angola.
Among those HIVDR-TS sequences, 46% were subtype C,
29% CRF01_AE, 7% subtype A and 10.5% untypeable.
To explore the difference of ambiguous mutation between
subtypes, we collected a dataset of 63 subtype B sequences
Figure 2. Distribution of ambiguous mutations and data statistical description of three data subsets. Sequence frequency
distribution with number of ambiguous mutations (AMs) was plotted by subset: (A). Threshold (n=346), (B). Baseline (n=271), and
(C). Vietnam (VN) (n=68); and the statistical description of the 3 data subsets was plot (D) by number and index of ambiguous
mutations using the same method as described in Figure 1. The percentage in A-C indicated recent infections in a dataset classified
by having ≤2 AMs per sequence (indicated by dash line).
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generated from specimens collected people infected with HIV-1
within 155 days , and compared them with all the non-B
subtype or stratified non-subtype B sequences. These stratified
sequences were selected from the dominant subtype(s) in the
datasets, including subtype C from Botswana and Malawi,
subtype A from Kenya and Tanzania, and CRF01_AE from
China. Statistical analysis indicated that no significant
difference of ambiguous mutation was found between subtype
B and non-B subtypes (p=0.16) or subtype C (p=0.107);
however, significant differences were noticed between
subtypes B and A (p=0.001) or between subtype B and
CRF01_AE (p=0.011, Table 3, Figure 1-insert).
We couldn’t perform analysis of ambiguous mutation
preference on the basis of infection route due to the incomplete
infection route data from the China and Vietnam HIVDR-TS
and the Nigeria baseline monitoring survey.
Detection of HIV recent infections is challenging and crucial
for accurate HIV incidence and t-HIVDR estimations. We
pursued an investigational molecular approach using
ambiguous mutation for determining HIV infection status and
monitoring early development of HIVDR. We characterized
ambiguous mutations in recent (HIVDR-TS) and established
Figure 3. Proportional distribution of mutated and ambiguous mutated amino acids at HIVDR sites. The mutation score at
each of the drug resistance sites  was proportionally calculated with the mutated and ambiguous mutated amino acids for all the
sequences in the datasets. A mutated or ambiguous mutated amino acid was defined as an amino acid had mutated from a wild
type to a pure non-synonymous mutation or an ambiguous mutation in the mixture allele. The scores were summed by 1 for a pure
amino acid mutation and 0.5 for an ambiguous amino acid mutation, and then converted to percentages against the total number of
wild-type amino acids at the site. The distribution of drug resistance mutation scores was plot by the dataset of Threshold (bottom
panel), Vietnam (VN, central panel) and Baseline (top panel). The x-axis is the wild-type amino acids at drug resistance sites; the y-
axis is the drug resistance mutation score (%). The sites with obvious score changes across the 3 datasets from bottom to top panel
were labeled by up-triangle (increased), rhombus (remained), and down- triangle (decreased). Amino acids of protease gene (Prt)
were top-dash lined, and of reverse transcriptase gene (RT) were top-solid lined.
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infections (HIVDR baseline), and demonstrated the lower
ambiguous mutation index was associated significantly with
recent infections. We defined 2.04×10-3 ambiguous mutations/
site as a measure for infections within one year, referred as
recent infection in the current study. With the substitution rate
defined, the proportion of subjects: 75.2% in the threshold,
3.3% in the baseline, and 10.3% in the Vietnam dataset, was
classified being recent infections. These results provided data
on the accuracy of defining HIV-1 recent infections using the
WHO epidemiological criteria.
The dataset from Nigerian HIVDR monitoring baseline
survey representing established infections exhibited a
significantly higher mutation index (14.40×10-3 nts/site), which
clearly differentiated them from the recent infections in the
threshold subset, and served as a great calibrator for
determining the ambiguous mutation index for recent
infections. Based on our analyses, HIV infection status could
range from 1 to 10 years for the majority of subjects in the
Nigeria dataset, which reflects the reality because the subjects
enrolled for ART included established and chronic infections
according to the Nigeria HIV treatment guidelines [26,33]. The
distribution curve of ambiguous mutations reflects the
distribution of infection status of subjects in the dataset. For
instance, a narrow sharp curve indicates a uniformity of
subjects who had been infected around the same period of
time whereas a wide curve indicates a wider range of infection
status from recent to chronic infections. These could serve as a
tool to evaluate uniformity of infection status in subjects from a
dataset or cohort.
Although the Vietnam dataset was also from HIVDR-TS, only
10.3% of the subjects could be identified for being recently
infected. The overall high ambiguous mutation index
Table 3. Comparison of ambiguous mutation (AM) in the
recent infections of subtype B and other subtypes.
AM index (×10-3 nts/
site) 95% CI P-value
Non-B subtypes 346 2.03 1.62-2.21 0.16
A 33 2.55 1.59-3.52 0.001
B 63 1.71 1.01-2.40
C 183 1.40 0.99-1.81 0.107
CRF01_AE 20 3.26 2.02-4.50 0.011
Figure 4. Index of mutated and ambiguous mutated amino acids at HIVDR sites by data subset. The total score of drug
resistance mutations caused by pure mutated amino acids or by ambiguous mutated amino acids was calculated separately for
each of the data subsets, and divided by the number of total drug resistance sites  to obtain the index of mutated or ambiguous
mutated amino acids by subset. The definition and score calculation of pure mutated and ambiguous mutated amino acids were
described in Figure 3.
Estimating HIV Recent Infection & Drug Resistance
PLOS ONE | www.plosone.org 8 October 2013 | Volume 8 | Issue 10 | e77649
(18.32×10-3 nts/site) similar to the one found in the Nigeria
baseline dataset was somewhat not surprising because in
Vietnam the two highest HIV risk groups were IDUs and
commercial sex workers [39,42] and the subjects in the
Vietnam HIVDR-TS were enrolled at VCT [28,30]. In contrast,
those from other HIVDR-TS surveys except for China were
enrolled at ANC clinics. They were women attending their first
pregnant visits and most likely infected through heterosexual
transmission [27,29,31,32]. Because the mechanism of HIV
evolution is transmission route dependent [2,21], IDUs have
only about 40% of the chance being infected with a single
virion and transmission of multi-viral strains through injection
would amplify the founder effect in folds, leading to higher
genetic diversities [20,43,44]. These might explain the Vietnam
dataset and the two subjects with outlier sequences from China
(6 IDUs were identified in the dataset, personal
communication) and Angola. Thus, those with higher
ambiguous mutations specifically in Vietnam dataset might not
be established infections, but IDUs.
Emergence and transmission of HIVDR is an on-going
concern in the scale-up ART programs in resource-limited
settings. To increase the elements of HIVDR surveillance and
monitoring, we utilized the ambiguous mutation approach to
monitor early emerged DR mutations and distinguish them from
fitted DR mutations. Our data revealed an increase trend in DR
mutations caused by ambiguous mutations (0.543.48×10-2) but
a constant level of fitted DR mutations (7.50-7.89×10-2) from
recent to established infections, indicating that t-HIVDR
background exists in ART-naïve populations in which new
HIVDR mutations continue to emerge over time. We also
identified multiple DR sites that had mutational scores
increased, remained stable or deceased over the time of
infections. This dynamic distribution profile may be valuable for
predicting early development of possible DRs in HIV-infected
populations which provide data to decision maker for regimen
considerations and/or selections.
Utilization of ambiguous mutations for predicting time of HIV
infections may represent a relatively new sensitive approach.
There are limited data available which were mainly focused on
subtype B sequences, including two publications on HIV pol-
gene [23-25] and one on env-gene . The analyses focused
on pol-gene found that >0.45-0.5% of ambiguous nucleotides
provided strong evidence against a recent infection within 1
year and that ambiguous mutation rate constantly increased by
0.2% per year for the first 8 years of HIV infections. Our finding
in which recent infections were defined as having an
ambiguous mutation rate of 2.03×10-3 nts/site/year is in
agreement with these studies and this is also corroborated with
the substitution rate of 2×10-3 nts/site/year as reported by
Our observation on subtype preference in viral ambiguity
showed no significant difference between subtype B and
overall non-B subtypes (p=0.16), which is consistent with a
recent study . However, our subtype-stratified analyses
appear to show somewhat different pictures since subtype A
(p=0.001) and CRF01-AE (p=0.011) did show higher ambiguity
when they were compared to subtype B. We believe that the
discrepant results may be attributed to the smaller sample size
that we have for these subtypes (Table 3). In this study, we
classified 75.2% of subjects in the threshold surveys were
infected within one year and this is in agreement with a recent
study in which 73% of the pregnant women were identified as
recent infections within one year from the datasets generated
using the same WHO epidemiological criteria for identifying
recent infections in resource-limited countries .
However, there are limitations in our study. We characterized
ambiguous mutations with datasets based on recent and
established infections. However, due to the limited
epidemiological data, we couldn’t conduct analysis and
interpret the data based on transmission modes. We could only
confirm the heterosexual transmission route for the women
recruited at ANC clinics because they were all at their first
pregnancy and diagnosed the first time with HIV infections.
Therefore, the founder effect would likely be the viral replication
mechanism, and the ambiguous mutation detected in the
HIVDR-TS datasets by Sanger sequencing would reflect the
genetic diversity in the viral population. However, for the
dataset with suspected IDUs, Sanger sequencing might not be
able to resolve the genetic diversity due to the multi-virion
infection nature. With the progress on new generation
sequencing technologies, e.g. deep or single genome
sequencing, the multi-virion infection could be resolved at
individual variant level to reflect the true genetic diversity
[44-46]. We identified significant higher ambiguous mutations
occurred in subtype A and CRF01_AE based on relatively
small sample size. Studies on a larger number of these
subtypes using epidemiologically defined cohorts of HIV
infections would be useful to confirm our findings and further
our understanding of ambiguous mutation preferences. Lastly,
we detected an increase of ambiguous mutations at HIVDR
sites. Due to the lack of clinical data, we couldn’t determine a
threshold of early developed minor HIVDR mutations that
would have clinical significance for treatment and regimen
In summary, we characterized ambiguous mutations in HIV-1
protease and reverse transcriptase gene regions with likely
recent and established infections. We defined an ambiguous
mutation index for detecting HIV recent infections and
characterized the distribution of ambiguous mutations for
monitoring the early development of HIVDR. Our data suggest
that molecular characterization of ambiguous mutations in
HIV-1 may serve as an additional tool along with serologic
assays to differentiate recent from established infections,
evaluate infection status, and monitor the early development of
We thank our colleagues in the countries of Angola, Botswana,
China, Kenya, Malawi, Tanzania and Vietnam for their support
and contributions to the HIVDR surveys, and members in the
Drug Resistance and Molecular Bioinformatics Team for
generating the sequence data and technical support.
Dr. Duc B. Nguyen received training support from Emory AIDS
International Training and Research Program (NIH/FIC D43
Estimating HIV Recent Infection & Drug Resistance
PLOS ONE | www.plosone.org 9 October 2013 | Volume 8 | Issue 10 | e77649
Disclaimer: Use of trade names is for identification only and
does not constitute endorsement by the U.S. Department of
Health and Human Services, the Public Health Service, or the
Centers for Disease Control and Prevention. The findings and
conclusions in this paper are those of the authors and do not
necessarily represent the views of the Centers for Disease
Control and Prevention.
Conceived and designed the experiments: DPZ CY. Performed
the experiments: MR EB DBN KD JRD. Analyzed the data:
DPZ. Contributed reagents/materials/analysis tools: MR EB
DBN CY JNN. Wrote the manuscript: DPZ CY JNN.
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