1. Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Routine HIV-1 genotyping as a tool to identify
dual infections
Marion Cornelissena
, Suzanne Jurriaansa
, Karolina Kozaczynskaa
,
Jan M. Prinsb
, Raditijo A. Hamidjajaa
, Fokla Zorgdragera
,
Margreet Bakkera
, Nicole Backa
and
Antoinette C. van der Kuyla
Objectives: The incidence of HIV-1 dual infections is generally thought to be low, but
as dual infections have been associated with accelerated disease progression, its
recognition is clinically important. Methods to identify HIV-1 dual infections are time
consuming and are not routinely performed.
Design: Genotyping of the HIV-1 protease and reverse transcriptase (prot/RT) genes is
commonly performed in the western world to detect drug-resistance mutations in
clinical isolates. In our hospital, prot/RT baseline sequencing is part of the patient care
for all newly infected patients in the Amsterdam region since 2003. We reasoned that
degenerate base codes in this sequence could indicate either extensive viral evolution
or infection with multiple HIV-1 strains.
Methods: We amplified, cloned and sequenced multiple HIV-1 envelope (env)-V3 and
gag sequences from patients with 34 or more (range 34–99) degenerate base codes in
the ViroSeq genotyping RT sequence (37 out of 1661 available records) to estimate the
number of HIV-1 dual infections in this group.
Results: Of the 37 patients included in this study, 16 (43.2%, equal to 1% of the 1661
total records) had an HIV-1 dual infection based on phylogenetic analysis of env-V3/gag
sequences. If only sequences with 45 or more degenerate base codes were taken into
account, 73.3% of patients showed evidence of a dual infection.
Conclusion: We describe an additional use of routinely performed HIV-1 genotyping.
In patients with a high number of degenerate bases (! 34) in RT it is important to
consider the possibility of a dual HIV-1 infection.
ß 2007 Lippincott Williams & Wilkins
AIDS 2007, 21:807–811
Keywords: Degenerate base codes, drug resistance, dual infection, env sequence
analysis, HIV-1, ViroSeq
Introduction
Reports of HIV-1 dual infections in individual patients
have been published since the early 1990s [1], but
screening of larger cohorts suggested that the incidence of
multiple HIV-1 infections is mostly low [2–5], but
not always [6,7]. HIV-1 dual infections are divided into
co-infections (occurring before a specific HIV immune
From the a
Laboratory of Experimental Virology, Department of Medical Microbiology, Centre for Infection and Immunity
Amsterdam, Amsterdam, the Netherlands, and the b
Department of Internal Medicine, Division of Infectious Diseases, Tropical
Medicine and AIDS, Academic Medical Centre of the University of Amsterdam, Amsterdam, the Netherlands.
Correspondence to Antoinette C. van der Kuyl, Laboratory of Experimental Virology, Department of Medical Microbiology, Centre
for Infection and Immunity Amsterdam (CINIMA), Academic Medical Centre of the University of Amsterdam, Meibergdreef 15,
1105 AZ Amsterdam, the Netherlands.
Tel: +31 20 5666778; fax: +31 20 5669064; e-mail: a.c.vanderkuyl@amc.uva.nl
Received: 1 June 2006; revised: 24 August 2006; accepted: 6 September 2006.
ISSN 0269-9370 Q 2007 Lippincott Williams & Wilkins 807

2. Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
response has been established), and superinfections
(in which a second virus successfully enters the host
after seroconversion) [8]. HIV-1 dual infections are seen
as problematic as they can give rise to recombinant viruses
with novel properties, have instigated doubts about the
efficacy of vaccine protection, and have been correlated
with disease progression [1,8]. Whether in a single
patient or in a larger cohort, however, it is notoriously
difficult to detect HIV-1 dual infections. Not only are the
methods, polymerase chain reaction amplification and
heteroduplex mobility assay or sequencing, relatively
time consuming, but also the sampling timepoints need
to be chosen carefully. Both dramatic fluctuations of
viral sequences in serum [9–11], or the outgrowth of
one virus or a novel recombinant [12–15] have been seen
in dual infections, suggesting that double infections can
easily be missed when analysing a single sample. We
report that the HIV-1 reverse transcriptase (RT) sequence
routinely requested by treating physicians to assess viral
drug-resistance mutations in patients failing antiretroviral
therapy offers an additional way of detecting HIV-1 dual
infections. In this sequence, the amount of degenerate
base codes reported can be an indication of extensive virus
evolution, but can also point to the presence of multiple
virus strains. In our analysis of 37 patients with a
degenerate base count of 34 or more (range 34–99) in the
ViroSeq RT sequence, 16 were found to have an HIV-1
dual infection.
Methods
Patients
HIV-1 protease/reverse transcriptase (prot/RT) gene
sequences, encompassing the complete protease sequence
and the first 335 codons of the RT gene, are routinely
generated in our hospital with the ViroSeq HIV-1
genotyping kit version 2 (Celera Diagnostics, Alameda,
California, USA). Electrophoresis and data collection are
performed on an ABI PRISM 3100 genetic analyser
(Applied Biosystems, Foster City, California, USA) [16].
A total of 1661 prot/RT gene sequence records were
available for our study, obtained from 1319 HIV-infected
patients. Most prot/RT sequences were generated
because of therapy failure, but since 2003 the ViroSeq
prot/RT sequence is part of the standard patient care for
every newly HIV-1-infected patient from the Amsterdam
region of the Netherlands and is determined at their first
hospital visit. Therefore, approximately 400 of the
1661 prot/RT gene records are so-called baseline
sequences that are not associated with therapy failure.
A total of 37 sequence records were selected for further
analysis based arbitrarily upon a degenerate base count of
34 or more in the RT part of the sequence. Degenerate
base codes (single-letter nucleotide codes of the
International Union of Biochemistry) taken into account
are R (A or G), Y (C or T), K (G or T), M (A or C),
S (G or C), W (A or T), B (C, G or T), D (A, G or T),
H (A, C or T), and V (A, C or G). H, Vor N (any base)
were never detected. Degenerate positions were
randomly distributed in RT, with no special emphasis
on known drug-resistance positions. The selection was
found to contain a patient described earlier as having an
HIV-1 triple infection [14]. This patient was not
sequenced again, but was included in the results.
Analysis of HIV-1 env-V3 and gag sequences
The V3 sequence of the HIV-1 envelope gene was
amplified as described [17] from the selected patient
samples. V3 fragments were cloned with the TOPO TA
cloning kit (Invitrogen, Carlsbad, California, USA), and
16 clones per patient were selected and sequenced.
Sequences were aligned with reference HIV-1 V3
sequences (from the Los Alamos National Laboratory at
http://hiv-web.lanl.gov) using ClustalW available in
BioEdit Sequence Alignment Editor version 7.0.1
(www.mbio.ncsu.edu/BioEdit/bioedit.html). Neigh-
bour-joining (NJ) trees based upon Kimura two-
parameter distances were constructed with the
MEGA software package (www.megasoftware.net),
and 1000 bootstrap replicates were analysed. Additional
phylogenetic analyses were performed with the parallel
version of MrBayes 3.1 (http://mrbayes.net), run at the
SARA High Performance Computing facilities
(www.sara.nl). SARA modified the program so that it
uses the sprng library (http://sprng.cs.fsu.edu/) to
generate independent streams of random numbers in
the parallel processes.
For 13 patients, further analyses were performed using an
amplified and cloned 720 nucleotide HIV-1 gag gene
fragment [18], encompassing most of p17 and the first
part of p24, in a similar approach as described above to
confirm the presence or absence of divergent sequence
clusters.
Definition of dual infection
The detection of HIV-1 dual infections was based upon
phylogenetic analyses of both nucleotide and amino
acid sequences of env-V3 or gag fragments with at
least 15 cloned sequences per patient. Positive identi-
fication was based upon the following criteria that
should be true with both the NJ method and
with Bayesian inference of phylogeny: sequences of a
single patient cluster independently, or sequences of
a patient cluster together, but the bootstrap/posterior
probability value connecting the branches is low (values
under 80/0.8 are here considered insignificant). In many
patients divergent sets of sequences were found that
nevertheless clustered together with high confidence
levels, and this was always attributed to viral evolution
and not dual infection. A representative NJ tree of
HIV-1 subtype B env-V3 sequences of five patients is
shown in Figure 1.
808 AIDS 2007, Vol 21 No 7

3. Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Results
The V3 fragment of the HIV-1 envelope gene was
amplified, cloned and sequenced for 36 patients with
a degenerate base count of 34 or more in the ViroSeq
HIV-1 RT sequence. For another patient, V3 sequences
were already available [14]. The results are summarized in
Table 1. Sixteen patients were found to have an HIV-1
dual infection based upon phylogenetic analysis of the
cloned V3 and gag sequences (43.2% of patient records
analysed, equal to 1% of the total 1661 genotyping
records). Both nucleotide and translated amino acid
sequences were analysed. In 12 cases, the patient’s plasma
contained sequences that formed at least two distinct
clusters, separated by reference sequences or by sequences
from other patients. In the four other cases, sequences
clustered together but with low, insignificant bootstrap
values (range 35–73) in both trees. Eleven of the dual
infections (68.8%) occurred with B subtypes, three dual
infections (18.8%) were with non-B subtypes (AG/AG,
D/D, and C/C, respectively). A single mixed infection
with subtype B and subtype CRF01_AE was found, apart
from the triple infected patient described earlier [14],
who was infected with CRF01_AE and two subtype B
viruses. The non-B/non-AE dual infections were all in
patients of African descent. Of the subtype B dual
infections, two patients were of unknown origin, eight
were from the Netherlands, and one was from Israel. Both
patients with B/AE dual infections were Dutch.
Discussion
Our study shows that a high degenerate base count in the
ViroSeq HIV-1 RT sequence is an excellent indicator of
an HIV-1 dual infection. We have analysed V3 and gag
sequences of 37 patients with a degenerate base count in
RTof 34 and more, and found support for a dual infection
in 16 of them (43.2%). If we limit the analysis to those
with a degenerate base count of 45 and over, 73.3% of
the patients show evidence of an HIV-1 dual infection
(11/15). In total, however, HIV-1 dual infections are rare:
of the 1661 ViroSeq RT sequences available, only 37 had
a degenerate base count of 34 or more (2.2%), of which
16 had an HIV-1 dual infection (1%).
In most cases, the treating physician ordered the
genotyping because of suspected drug resistance.
Approximately a quarter of the sequences available were
Detecting HIV-1 dual infections Cornelissen et al. 809
R05-14090
R9606590
#
#
#
#
#
#
R00-08950
R05-14090
subtype B-BR30
subtype B-BR04
#
#
#
#
#
#
#
#
#
R00-08950
subtype B-BR03
subtype B-BR28
R9606590
$
$
$
$
$
$
$
$
$
$
$
$
$
$
$
R04-15734
R05-19117
LBV217
SE6165
JP88
JV83
subtype G
100
100
99
100
96
99
98
92
100
47
99
0.05
Fig. 1. Representative neighbour-joining tree based upon
Kimura two-parameter distances of HIV-1 env-V3 subtype
B nucleotide sequences. Four subtype B reference sequences
were also included in the analysis, and four subtype G
sequences served as outgroup. Patient R05-19117 (*) is an
example of a patient showing virus evolution, but no dual
Fig. 1. (Continued ).
infection (high bootstrap value). Dual infections are revealed
by the sequences of patient R04-15734 ($), forming two
clades connected by a very low bootstrap value (here 47),
and the sequences of patient R05-14090 (^), R9606590 (&),
and R00-08950 (#) forming two distinct clades.

4. Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
so-called baseline sequences, however, determined at the
first hospital visit of a newly recognized HIV-1 infected
patient. It can be seen from Table 1 that most dual
infections were found in baseline sequences (75% of the
dual infections or 3% of the total available baseline
sequences), and less in sequences generated because of
suspected drug resistance (25% of the dual infections or
0.3% of total genotyping sequences ordered because of
therapy failure).
The highest RT degenerate base counts were found to
correspond to dual infections with different HIV-1
subtypes (Table 1). Genetic diversity is expected to be
larger there than in double infections with a more
similar virus, such as in within-subtype infections, or in
re-infections by the same partner. Re-infections are
estimated to have the lowest genetic diversity and could
represent the detection limit of dual infections by
phylogenetic analysis. Possibly, re-infections are
represented in our data set as showing low confidence
levels in the phylogenetic analysis. In many cases, such
infections will be missed because the newly infecting
strain cannot reliably be told apart from the earlier
strain.
Whether the novel dual infections reported here resulted
from HIV-1 co-infection or from HIV-1 superinfection
cannot be estimated from the data. In at least three
patients, however, one cluster of viral sequences showed
significantly less sequence variation than the other cluster,
suggestive of a recent superinfection.
This study shows that genotyping has an additional use in
detecting HIV dual infections, especially if baseline
sequence determination is part of the routine patient care.
It is important for physicians to consider the possibility of
a patient having a second HIV-1 infection, as this can
result in a (temporary) increase in the viral load or in
clinical symptoms, and is generally associated with disease
progression. Vice versa it is important to consider
the likelihood of an HIV-1 superinfection in an HIV-
1-infected patient presenting with an increase in viral load
or clinical illness, and routine genotyping could then help
in the diagnosis.
810 AIDS 2007, Vol 21 No 7
Table 1. HIV-1 dual infections in patients with 34 or more degenerate International Union of Biochemistry codes in the Viroseq RT sequence.
Patient no. Sex
Age
(years)
CD4 cell
count Â106
/ml
HIV-1
copies/ml
HIV-1
subtype Indication
Degenerate
base codes in RT
HIV-1dual
infection
R05-22667 M 58 Not determined 112 673 AE/B Baseline 99 Yes
R03-16410 M 40 310 1 779 260 AE/B/B Super-infection 85 Yes
R05-01201 M 31 Not determined 53 167 B Baseline 58 Yes
R05-12726 M 40 Not determined > 500 000 B Baseline 56 Yes
R03-17974 M 40 170 2664 A-like Therapy failure 55 No
R03-23836 M 36 170 60 612 AG Baseline 54 Yes
R05-14090 M 46 560 10 710 B Baseline 54 Yes
R04-15734 M 45 380 148 700 B Baseline 52 Yes
R02-20217 M 28 1050 8388 B Baseline 51 Yes
R03-09948 F 36 210 89 764 D Therapy failure 51 No
R05-16958 F 26 200 2119 D Therapy failure 51 Yes
R05-01866 M 45 380 70 454 B Baseline 50 No
R04-25021 M 42 100 26 502 C Baseline 48 Yes
R00-02189 M 50 Not determined 1200 B Therapy failure 46 No
R9606590 M 54 Not determined 31 000 B Pre-HAART 45 Yes
R00-09299 F 33 40 230 000 B Therapy failure 44 No
R04-20005 F 43 140 204 556 D Baseline 42 No
R04-26287 F 44 230 7651 AG Baseline 42 No
R03-04387 F 40 330 3706 AG Baseline 41 No
R04-24562 M 42 660 636 B Therapy failure 41 No
R05-19423 M 38 420 432 375 B Baseline 41 No
R9607901 M 41 Not determined Not determined B Baseline 41 No
R00-08950 M 42 780 4300 B Baseline 39 Yes
R03-14144 M 40 Not determined 180 000 B Baseline 39 Yes
R04-03289 F 36 140 1 971 240 B Baseline 39 No
R05-19117 F 56 180 74 585 B Baseline 39 No
R06-03030 M 44 10 161 603 B Pre-HAART 39 No
R9921479 M 50 390 29 000 B Baseline 38 Yes
R03-09815 F 33 50 196 079 AG Baseline 36 No
R02-04486 M 38 Not determined 110 000 B Baseline 35 No
R04-01278 M 55 Not determined Not determined B Therapy failure 35 Yes
R04-17261 M 38 Not determined Not determined B Baseline 35 Yes
R05-13079 M 30 120 57 481 B Therapy failure 35 No
R05-13441 M 38 20 98 703 AG Stop ART 35 No
R9608991 M 36 ND 37 000 B Pre-HAART 35 No
R03-20285 F 28 480 38 957 AG Baseline 34 No
R04-22370 M 39 Not determined Not determined B Therapy failure 34 No
ART, Antiretroviral therapy; RT, reverse transcriptase.

5. Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Acknowledgements
The authors would like to thank Dr Willem Vermin
(SARA, Amsterdam, the Netherlands) for modifying the
MrBayes program.
Conflicts of interest: None.
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