Increasing HIV-1 molecular complexity among men who have sex with men in Bangkok.

AIDS Research and Human Retroviruses Increasing HIV-1 Molecular Complexity among MSM in Bangkok (doi: 10.1089/AID.2014.0139) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

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1 Increasing HIV-1 Molecular Complexity among MSM in Bangkok

Wanna Leelawiwat a, Wiriya Rutvisuttinuntc, Miguel Arroyoc*, Famui Mueanpaia, Oranuch Kongpechsatit a, Wannee Chonwattana a, Supaporn Chaikummao a, Mark de Souzad, Frits van Griensvena,b, Janet M McNicholla,b, Marcel E Curlina,b

a

Thailand Ministry of Public Health – U.S. Centers for Disease Control and Prevention

Collaboration, Nonthaburi, Thailand; bDivisions of HIV/AIDS Prevention, Centers for Disease Control and Prevention, Atlanta, GA, USA; cDepartment of Retrovirology, Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand; d SEARCH Thailand, Thai Red Cross AIDS Research Center, Bangkok, Thailand *Present address: Miguel A.Arroyo, cDepartment of Pathology and Area Laboratory Services, Dwight David Eisenhower Army Medical Center, Augusta, GA, USA

Running head characters: 37 Running Head: Increasing HIV-1 Molecular Complexity Tables and Figures: 4

Disclaimer: The findings and conclusions in this article are those of the authors and do not necessarily represent the views of the U.S. Centers for Disease Control and Prevention or the official policy of the Department of Army, Department of Defense, or the U.S. Government. The authors have no conflicts of interest to disclose.


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2 Presented in part at: 1. 5th Conference on HIV Pathogenesis Treatment and Prevention, Cape Town, South Africa, July 19-22, 2009. Abstract number CDA053. Title: “High HIV-1 Genetic Complexity in Men Who Have Sex with Men (MSM) in Bangkok, Thailand” 2. 18th International AIDS Conference, Vienna, Austria, July 18-23, 2010. Abstract number WePe0010. Title: “High HIV-1 Genetic Complexity in Men Who Have Sex with Men (MSM) in Bangkok, Thailand” 3. AIDS Vaccine 2011, Bangkok, Thailand, September 12-15, 2011. Abstract number P20.04. Title “Characterization of HIV-1 Subtype Distribution among Thai MSM using MHAbce, a High Throughput Approach for Molecular Epidemiology Studies” 4. 20th Conference on Retroviruses and Opportunistic Infections, Atlanta, GA, USA, March 3-6, 2013. Abstract number I-120. Title “HIV-1 subtype and disease progression in seroincident HIV infections among MSM in Thailand”


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3 Abstract: Background: In Thailand, new HIV-1 infections are largely concentrated in certain risk groups such as men who have sex with men (MSM), where annual incidence may be as high as 12% per year. The paucity of information on the molecular epidemiology of HIV-1 in Thai MSM limits progress in understanding the epidemic and developing new prevention methods. We evaluated HIV-1 subtypes in seroincident and seroprevalent HIV-1 infected men enrolled in the Bangkok MSM Cohort Study (BMCS) between 2006 and 2011. Methods: We characterized HIV-1 subtype in 231 seroprevalent and 194 seroincident subjects using the multihybridization assay (MHA). Apparent dual infections, recombinant strains, and isolates found to be non-typeable by MHA were further characterized by targeted genomic sequencing. Results: Most subjects were infected with HIV-1 CRF01_AE (82%), followed by infections with recombinants (11%, primarily CRF01_AE/B recombinants), subtype B (5%), and dual infections (2%). More than 11 distinct chimeric patterns were observed among CRF01B_AE/B recombinants, most involving recombination within integrase. A significant increase in the proportion of non-typeable strains was observed among seroincident MSM between 2006 and 2011. Conclusion: CRF01_AE and subtype B were the most and least common infecting strains, respectively. The predominance of CRF01_AE among HIV-1 infections in Thai MSM participating in the BMCS parallels trends observed in Thai heterosexuals and injecting drug users. The presence of complex recombinants, and a significant rise in non-typeable strains suggest ongoing changes in the genetic makeup of the HIV-1 epidemic in Thailand, which may pose challenges for HIV-1 prevention efforts and vaccine development.


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4

Word count: 250

Key words: Men who have sex with men (MSM), HIV-1, subtype, Molecular, Epidemiology,

Thailand.


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5 1

Introduction

2

Despite significant progress in curbing the expansion of the HIV-1 epidemic on a global level 1,

3

men who have sex with men (MSM) continue to be disproportionately affected by HIV-1

4

infection in the majority of industrialized nations, and middle and lower income countries 2-5. In

5

several regions of Asia, the HIV-1 prevalence among MSM has more than doubled between

6

2006 and 2011 5-7. In Thailand, high HIV-1 prevalence and incidence continue to be reported in

7

this population, and it is estimated that approximately 30% of new infections occur in

8

predominantly young MSM 8-11. The latter group appears to be at particularly high risk of HIV-1

9

acquisition, with rates as high as 12.2 per 100 person-years as reported recently among 15-21

10

year old MSM who came in for HIV voluntary testing and counseling in Bangkok 9, 12.

11 12

Because of variability of the HIV-1 genome, the distribution of viral genetic polymorphisms may

13

vary significantly between different geographical regions, and between different risk groups

14

within the same area. This may reflect founding viral strains at the time of introduction,

15

diversification over time, and behavioral factors determining cross-exposure between risk groups

16

13-16

17

for the development of effective HIV-1 prophylactic vaccines 14, 15, 17 designed to elicit cytotoxic

18

T lymphocyte responses to relevant epitopes 18-21, or antibodies capable of neutralizing a broad

19

range of possible infecting viruses 22-24. Molecular epidemiological studies are an important tool

20

to understanding patterns of transmission from region to region, defining transmission pathways

21

between groups and individuals, and guiding development of interventions capable of eliciting

22

protective adaptive immune responses.

23

. Knowledge of circulating HIV-1 viral strains and subtypes is also considered fundamental


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6 1

Despite the potentially informative nature of viral genetic studies, compared to other risk groups

2

in Thailand 25-32, the subtype distribution of HIV-1 strains prevalent among MSM has not been

3

well characterized. Only one previous study identified 99 MSM from among many clients

4

attending an HIV-1 voluntary counseling and testing clinic in Bangkok 33. While limited due to

5

its cross-sectional nature and possible inclusion of repeat-clients, the authors found a higher

6

prevalence of non-CRF01_AE infections in MSM compared to other risk groups. The data also

7

suggested that some MSM may have had dual infection, but this was not definitively evaluated

8

using gene sequencing methods.

9 10

Given the increasing importance of HIV-1 transmission among MSM in sustaining the overall

11

HIV-1 epidemic in Thailand and on a global level, it is vital to develop a clear picture of the

12

molecular epidemiology of HIV-1 infection in MSM, and understand the significance of regional

13

and temporal trends in this group. Though typically more resource-intensive and challenging to

14

implement, cohort studies distinguish themselves from cross-sectional studies in providing an

15

opportunity to evaluate HIV-1 genetic epidemiology in a well-characterized population over

16

time. We previously established an MSM cohort study in Bangkok, Thailand 9. Enrollment began

17

in 2006 and accrued 1744 participants. Over 5 years of follow-up (2006-2011), we documented a

18

high HIV-1 prevalence (22.4%) and incidence (5.9/100 person-years) in this population 9. In this

19

paper, we characterize seroprevalent and seroincident infecting HIV-1 subtypes by multi-region

20

hybridization assay (MHAbce) in this cohort. We further evaluate possible dual and recombinant

21

infections by gene sequencing, and present trends over time among sero-prevalent and

22

seroincident cases over five years of follow-up.

23


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Methods

2

Study subjects and specimen collection

3

Screening and enrollment of participants in the Bangkok MSM Cohort Study (BMCS) was

4

conducted from April 2006 to January 2008 (period 1), and from September 2009 to November

5

2010 (period 2) 9. The present viral diversity study includes seroprevalent study subjects

6

enrolled during period 1, and seroincident study subjects identified during follow-up of men

7

enrolled during both periods until December 2011. Oral fluid, EDTA whole blood, and citrate

8

plasma were collected from all participants at baseline and follow-up visits every four months.

9

Blood specimens for MHA, CD4, and HIV-1 RNA viral load were collected at the time of

10

enrollment from seroprevalent study subjects, and from seroincident study subjects on the date of

11

the first HIV-1 seropositive test or shortly thereafter.

12 13

Ethical Review

14

The protocol of this study was reviewed and approved by the Thailand Ministry of Public Health

15

Ethical Review Committee for Human Subjects Research, the Institutional Review Board of the

16

U.S. Centers for Disease Control and Prevention, and the Human Subject Research Review

17

Board of the U.S. Army Medical Research and Materiel Command. Informed consent was

18

obtained from all participants prior to enrollment in the study.

19 20

HIV-1 Testing

21

Subjects were screened for the presence of HIV-1 antibodies in oral fluid (OraQuick, Orasure

22

Technologies, USA) at enrollment and every four months thereafter. HIV-1 reactive oral fluid

23

tests were confirmed in blood using three consecutive HIV-1 rapid tests in accordance with Thai


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National Guidelines for rapid HIV testing (Determine, Abbott, USA; DoubleCheck, Orgenics

2

Ltd., Israel, or SD Bioline, Standard Diagnostics, Inc, Korea; and Capillus HIV-1/2, Trinity

3

Biotech, USA, or HIV1/2 Core, Core Diagnostic, UK) 9. From February 2010 onwards, plasma

4

samples from volunteers testing non-reactive on oral fluid were evaluated for acute HIV-1

5

infection using 4th generation EIA (AxSym HIV 1/2 Ag/Ab Combo, Abbott, USA) and NAT

6

(Aptima Genprobe, USA). Study subjects were considered seroprevalent if determined HIV-1

7

infected at the time of enrollment, and seroincident if found HIV-1 infected during follow-up.

8 9

CD4+ cell count and plasma HIV-1 RNA viral load determination

10

CD4+ cell count was performed on EDTA whole blood by single platform volumetric flow

11

cytometry (Guava Easy CD4, Millipore, USA). HIV-1 RNA was quantified in plasma using

12

COBAS TaqMan HIV-1 version 1.0 (Roche Molecular Systems, USA). The lower limit of

13

detection was 47 copies/mL. Plasma without HIV-1 RNA detected was recorded as 0 copy/mL.

14 15

HIV-1 Genotyping by MHA

16

HIV-1 RNA was extracted from 200 µL of plasma using the QIAamp Viral RNA Mini Kit

17

(Qiagen, USA). HIV-1 negative samples and water were used as negative controls, and

18

previously characterized HIV-1 positive samples were used as positive controls. HIV-1

19

genotyping was performed by multi-region hybridization (MHAbce version 2), optimized for the

20

detection of HIV-1 subtype B (referred to as Western B strain), subtype C and CRF01_AE 34,

21

using an ABI HT 7900 real-time PCR machine (Applied Biosystems, USA). Classification of

22

circulating HIV-1 subtypes by MHAbce was identified according to established criteria 33. A

23

genotype was assigned when probe hybridization occurred in at least four of eight genomic


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regions: 1) a single subtype (B, C, or CRF01_AE) was assigned when all hybridizing probes

2

were of the same subtype; 2) recombinant forms (BE, BC, CE, or BCE) were assigned when two

3

or more different subtype probes hybridized in different regions of the genome; and 3) infection

4

with multiple subtypes (for example putative dual infection) was tentatively assigned when

5

probes of two or more different subtypes hybridized in the same genome region (more than 1

6

subtype/region). We defined a strain as “non-typeable (NT)” when there was probe reactivity

7

and/or sequence information in fewer than four genomic regions, and “non-amplifiable (NA)”

8

when there was no evidence of PCR amplicons as determined by SyberGreen, a fluorescent dye

9

staining double-stranded DNA.

10 11

All putative multiple infections identified by MHAbce were confirmed by cloning of PCR-

12

amplified nucleic acids, followed by repeated MHAbce on individual clones. Briefly, PCR

13

amplicons corresponding to regions showing dual probe reactivity in MHAbce were ligated into

14

TOPO vector (TOPO TA cloning, Invitrogen, USA) carrying plasmid encoding antibiotic

15

resistance, and then transformed into competent cells. Transformants carrying ligated plasmids

16

were selected on LB plates containing 50 µg/mL Kanamycin and -galactose. Subsequently, 16-

17

32 ligated clones were used for repeated genotyping by MHAbce and for sequencing 35, as

18

specified below.

19 20

Genotyping by targeted genomic sequencing

21

All samples classified as “putative dual infections” and seroincident samples classified as

22

“recombinant” and “non-typeable” by MHAbce were further characterized by targeted genomic

23

sequencing. For apparent dual infections by MHAbce, 2-12 transformed clones were used for


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sequencing. For samples designated as “recombinant”, amplicons from all gene regions showing

2

possible recombination by MHA were used for bulk sequencing. For samples designated as

3

“non-typeable” by MHAbce, amplified DNA from all gene regions without MHA results were

4

used for bulk sequencing. All sequencing was performed with Big Dye terminators on an ABI

5

3130 Capillary sequencer (Applied Biosystems, USA) as previously described 34. Target gene

6

sequences from the HIV-1, rt, int, tat, gp120, gp41, nef were aligned with reference strains from

7

the Los Alamos HIV-1 database, including representative HIV-1 CRF01_AE, subtype B, subtype

8

C strains and recombinant strains circulating in Thailand and neighboring countries. Alignments

9

were made using Clustal W 36, and manually edited using Genetic Data Environment (GDE 2.4,

10

Rockville, USA) or MacClade 4.08a. Maximum-likelihood phylogenetic analyses were

11

performed using the best-fit model of molecular evolution estimated by Moldeltest using the

12

Akaike Information Criterion (AIC) 37. Phylogenetic trees were reconstructed under the general

13

time reversible model of nucleotide substitution, with proportion of invariable sites and gamma

14

distribution rate heterogeneity (GTR+I+G) using PAUP* 38 within Geneious Pro5.5.6

15

(Biomatters). Bootstrap resampling was performed with 500 replicates. A bootstrap value of

16

>70% was considered to be evidence of monophylogeny39. Confirmed dual infections were

17

defined as the presence of two different sequence subtypes or circulating recombinant forms in

18

one sample.

19 20

Statistical Analyses

21

Differences in median age, CD4+, and viral load between seroprevalent and seroincident study

22

subjects were evaluated with Wilcoxon rank-sum test using SAS version 9.3 (SAS Institute,

23

Cary, NC, USA). We evaluated trends in HIV-1 subtype distributions over time using the chi-


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11 1

square test with StatCalc (Epi Info 7). P-values < 0.05 were considered statistically significant.

2

Non-typeable and non-amplifiable samples were excluded from the denominator in calculations

3

of HIV-1 subtype distributions.

4 5

Results

6

HIV-1 prevalence, incidence, and participant characteristics

7

Of the 1,292 men who enrolled during period 1, 290 (22.4%) tested positive for HIV-1 infection.

8

Of the 1,372 men testing negative for HIV-1 infection at enrollment during periods 1 and 2, 216

9

seroconverted for HIV-1 infection during follow-up until December 2011 (HIV-1 incidence: 5.9

10

per 100 person-years). Blood samples for laboratory testing were collected on the same day of

11

first HIV-1 seropositive testing (61% of all subjects), or as soon as possible thereafter (median

12

duration: 13 days; range 1-487 days). Median CD4 cell count was higher in seroincident men

13

(479; range 5-1,105 cells/µL) than seroprevalent men (424; range 28-1,712 cells/µL; p < 0.05).

14

Median HIV-1 RNA VL was also higher among seroincident men (79,600; range 0-85,600,000

15

copies/mL) than seroprevalent men (43,550; range 0-2,010,000 copies/mL; p < 0.05). There was

16

no significant difference with respect to median age between the two groups of men (26 years;

17

range 18-52 years).

18 19

Performance of MHAbce version 2

20

A total of 278 (95.9%) seroprevalent and 211 (97.7%) seroincident study subjects had samples

21

available for HIV-1 subtyping by MHAbce. Of these 489 samples, 425 (86.9%) were

22

successfully genotyped by MHAbce and sequencing. Among seroprevalent subjects, 83% could

23

be typed, 12% were successfully amplified but HIV-1 subtype could not be assigned (non-


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typable by MHAbce only), and 5% failed PCR amplification (NA) due to low HIV RNA VL.

2

Among 211 seroincident subjects, 92% could be typed, 6% were non-typeable (by MHAbce and

3

sequencing), and 2% failed PCR amplification (appendix, Table 1).

4 5

HIV-1 subtype distribution, patterns of HIV-1 recombination, and dual infections

6

The distribution of HIV-1 genotypes in seroprevalent and seroincident subjects is described in

7

Table 2. Overall, most MSM (82%) were infected with CRF01_AE. Non-CRF01_AE strains

8

accounted for 18% of infections, among which 11% were recombinants, 5% were subtype B, and

9

2% were dual infections. Recombinant infections were primarily CRF01_AE/B. Using MHAbce,

10

18 putative dual infections were detected. Of these, seven (three among incident infections and

11

four among prevalent infections) were confirmed to be dual infections by phylogenetic analysis

12

of gene sequences (Table 2 and Figure 1). In the remaining 11 cases, which did not meet criteria

13

for dual infections, phylogenies were characterized by relatively long-branch lengths reflecting

14

highly diverse populations (mean genetic diversity = 0.043; range 0.002-0.24; data not shown).

15 16

Among the 43 CRF01_AE/B recombinants identified, eleven distinct patterns of inter-subtype

17

recombination were observed, including eight possible novel forms. Five recombinants involved

18

substitution within the integrase gene (Figure 2) and accounted for the majority (33/43) of

19

recombinations observed. In each case, sequences consisted of a predominant CRF01_AE

20

background with one or more subtype B fragments in integrase with or without recombination in

21

other regions; int only (20/43, 47%), int and nef (2/43, 5%), int and rt (5/43, 12%), int, rt and tat

22

(2/43, 5%), int, rt and gp120 (3/43, 7%), and int, rt, tat and nef (1/43, 2%). The remaining

23

recombinants had subtype B fragments in other regions; rt (5/43, 12%), nef (2/43, 5%), gp120


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(1/43, 2%), gp41 and nef (1/43, 2%), and tat (1/43, 2%). Three recombinants involving subtype

2

C were identified by MHA. In all cases, these consisted of a subtype B background with a

3

subtype C integrase fragment (B/C recombinant), with or without CRF01_AE fragments in p17

4

and gp41 (B/C/CRF01_AE recombinant).

5 6

Trends in HIV-1 subtype distribution during 2006-2011

7

The distribution of identifiable HIV-1 subtypes was similar between seroprevalent and

8

seroincident subjects. CRF01_AE was the most common while subtype B was the least common

9

strain. Among seroprevalent infections, there was no significant temporal trend in subtype

10

distribution from 2006 to 2008 (p >0.05). There was likewise no significant change in subtype

11

distribution over five years of follow-up among seroincident subjects, although in each group

12

there was some fluctuation in strain prevalence from year to year. We noticed a significant rise in

13

the proportion of non-typeable strains among seroincident subjects, from 0% in 2006 to 11% in

14

2011 (p=0.02) (Table 2).

15 16

Discussion

17

Our study is the first to describe the molecular epidemiology of HIV-1 infection among Thai

18

MSM in detail, and to characterize changes in subtype distribution over time. In this study, we

19

report HIV-1 genotypes circulating among MSM in Bangkok from 2006 to 2011. The most

20

common infecting viral strain was CRF01_AE, followed by recombinant involving CRF01_AE,

21

subtype B and subtype C, HIV-1 subtype B, and dual infections. The distribution of viral strains

22

was similar between seroprevalent and seroincident HIV-1 infections in our population. Dual

23

infection was confirmed in 2% of study participants. In this study, we observed several


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14 1

previously unreported patterns of recombination between CRF01_AE and HIV-1 subtype B.

2

Lastly, we observed a significant increase in the proportion of non-typeable strains among HIV-1

3

seroincident subjects over the study period.

4 5

HIV-1 molecular genotyping has been conducted in Thai MSM in only one other study 33.

6

Arroyo et al., performed subtyping using MHAbce without confirmatory sequencing on

7

specimens collected from 99 MSM attending HIV voluntary counseling and testing services in

8

Bangkok between 2006 to2007 33. In these MSM, CRF01_AE was identified as the infecting

9

strain in 75%, subtype B in 7%, and CRF01_AE/subtype B recombinants in 15%. The

10

distribution of HIV-1 subtypes found in the present study are consistent with those of Arroyo et

11

al., and provide a robust contemporary confirmation of this pattern in seroprevalent and

12

seroincident MSM over an extensive sampling period. Both studies identified a high proportion

13

of complex recombinant forms, and we note a significant rise in the frequency of non-typeable

14

strains. Sequence analysis in non-typeable study subjects suggests that one reason for failure of

15

MHA to provide subtype classification is increasing divergence within probe binding regions

16

over time (data not shown).

17 18

In the present study, we observed at least eleven distinct intersubtype recombinants. Most of

19

these involved subtype B replacement within integrase on a CRF01_AE background, either

20

alone or in combination with additional replacements within nef, tat, gp120, and rt (Figure 2).

21

One recombinant had pattern similar to CRFs 55 (int, rt) while the others may be new unique

22

recombinant forms (URF). CRF01_AE is a widely circulating strain prevalent in Southeast Asia

23

generally thought to be a complex chimeric virus composed of subtype A and E parental


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15 1

subtypes, though this has been questioned 40. The first recombinant between CRF_01AE and

2

subtype B was identified in Thailand in 2003 (CRF15_01B), and was shown to be composed of a

3

predominant CRF01_AE background with an HIV-1 subtype B segment within the viral env

4

gene 41. Several other CRF01/B recombinants have since been noted in Thailand 32, 42 and other

5

parts of South East Asia including CRFs33, 34, 48, 51, 52, 53, and 54

6

(http://www.hiv.lanl.gov/content/sequence/HIV/CRFs/CRFs.html). The new recombinant forms

7

identified here will require further molecular characterization and epidemiologic investigation to

8

be fully classified as specific to the study participant or true CRFs circulating more widely in the

9

Thai HIV epidemic43.

10 11

This analysis offers several strengths over previous studies. Our data were derived from a large

12

and well-characterized longitudinal cohort study, including both seroprevalent and seroincident

13

infections, and we performed targeted viral genetic sequencing in all cases where results could

14

not be unambiguously determined by sequence-specific hybridization methods. This study

15

design eliminates the possibility of confounding due to repeat visits, and allows for robust

16

characterization of epidemiologic trends in our population. However, several limitations should

17

also be considered. The gold standard for characterizing the subtype origins of an individual

18

virus is full-length genomic sequencing but this technique is currently not practical for larger

19

studies. The probe-hybridization approach used here allows for high-throughput analysis but

20

might fail to detect viral strains present at very low frequency, or novel recombinant forms with

21

short recombinant segments falling outside of the genomic regions studied. Although MHAbce

22

has been designed to detect currently recognized viral strains circulating in Asia 34, some

23

specimens may have been misclassified due to non-specific probe binding or failure to classify


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16 1

variant B strains. In addition, our study participants may imperfectly represent regional MSM

2

populations due to self-selection leading to overrepresentation of men with characteristics

3

associated with the outcomes studied here. For example, clustered HIV transmission events

4

occurring within relatively closed local sexual networks may increase the apparent prevalence of

5

certain HIV-1 genotypes 44

6 7

During the early HIV-1 epidemic, HIV-1 subtype B’ predominated among IDUs 30, 45 whereas

8

CRF01_AE infection was characteristic among those likely to have been exposed through sexual

9

contact 26, 46. However, more recently an increasing number of infections with CRF01_AE and

10

recombinant forms has been noted in all populations, with an associated decline in the proportion

11

of subtype B infections 27-29, 31, 33, 47. Other reports in the region have suggested similar shifts in

12

favor of CRF01_AE over HIV-1 subtype B. In China, subtype B infections have declined

13

dramatically from 90% in 2006 to only 20% in 2009, with a concomitant rise in the proportion of

14

CRF01_AE from 4% to 50% 48-51. CRF01_AE also appears to have made a recent incursion into

15

Japan, where HIV-1 infection had been nearly uniformly due to HIV-1 subtype B 52. The reasons

16

for this shift are unclear but could be related to subtype-specific differences in viral load during

17

early infection 53or behavior differences between risk groups13. The results obtained in our study

18

suggest a continued trend towards a more complex epidemic with a rising number of

19

recombinant forms and loss of pure subtype B infections. If these trends continue, we may

20

anticipate a mature HIV-1 epidemic in the Asia-Pacific region in which CRF01_AE and complex

21

CRF01_AE /B have displaced subtype B. These shifts will be of significance to efforts to

22

develop regionally effective HIV prophylactic vaccines and other means of mitigating HIV

23

transmission through epitope-specific immune responses.


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17 1 2

Acknowledgments

3

The authors would like to thank the participants in this study, and acknowledge the support and

4

funding from the U.S. military HIV-1 Research, and the Henry M Jackson Foundation. We also

5

thank Viseth Ngauy, Vatcharin Assawadarachai, Kultida Poltavee, Hathairat Savadsuk, and

6

Suwittra Chaemchuen of the Armed Forces Research Institute of Medical Sciences, Thailand for

7

their support in this study; Sodsai Tovanabutra, Gustavo Kijak, Eric Sander-Buell, Morgane

8

Rolland, and Francine McCutcheon, and Jerome Kim of the US Military HIV Research

9

Program, for their technical and intellectual input; Jaray Tongtoyai, Atittaya Sangiamkittikul,

10

Punneeporn Wasinrapee, Natthaga Sakulploy, Kusuma Auethavoranan, and Wanna Suwanaphan

11

of the Thai MOPH US – CDC Collaboration (TUC) laboratory for processing and testing all the

12

samples; Sarika Pattanasin, Boonyos Raengsakulrach, and Chonticha Kittinunvorakoon for

13

helpful advice; and remaining members of our collaborative study group.

14 15

Author Disclosure Statement

16

No competing financial interests exist

17 18


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Correspondence and Reprint Requests to:


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Table 2 Distribution of HIV-1 Subtypes in MSM in Bangkok, 2006-2011

2

Sampling year

Samples genotyped (No. of typeable samples)a

Subtype detected (Percentage)b

CRF01_AE

B

B/CRF01_ AE RC

B/C or B/C/CRF01_AE RC

NTc

NAc

Dual infections (B/ CRF01_AE)

Seroprevalent samplesd

2006 2007 2008

70 (58) 196 (162) 12 (11)

47 (81%) 130 (80%) 10 (91%)

3 (5%) 7 (4%) 0

6 (10%) 20 (12%) 1 (9%)

1 (2%) 2 (1%) 0

1 (2%) 3 (2%) 0

10 (14%) 2 (3%) 23 (12%) 11 (6%) 1 (8%) 0

Subtotal Trend p-value

278 (231)

187 (81%) 0.86

10 (4%) 0.35

27 (12%) 1.00

3 (1%) 0.31

4 (2%) 0.50

34 (12%) 13 (5%) 0.40 0.90

2 (2) 24 (23) 42 (41) 45 (43) 41 (34) 57 (51)

1 (50%) 21 (91%) 33 (81%) 34 (79%) 26 (76%) 47 (92%)

1 (50%) 0 4 (10%) 3 (7%) 4 (12%) 1 (2%)

0 2 (9%) 3 (7%) 6 (14%) 3 (9%) 2 (4%)

0 0 0 0 0 0

0 0 1 (2%) 0 1 (3%) 1 (2%)

0 0 0 1 (4%) 1 (2%) 0 1 (2%) 1 (2%) 5 (12%) 2 (5%) 6 (11%) 0

211 (194)

162 (84%) 0.44

13 (7%) 0.37

16 (8%) 0.45

0 NA

3 (1%) 0.73

13 (6%) 4 (2%) 0.02 0.56

2006

72 (60)

48 (80%)

4 (6%)

6 (10%)

1 (2%)

1 (2%)

10 (14%) 2 (3%)

2007

220 (185)

151 (82%)

7 (4%)

22 (12%)

2 (1%)

3 (1%)

23 (11%) 12(5%)

2008

54 (52)

43 (83%)

4 (8%)

4 (8%)

0

1 (2%)

2 (4%)

0

2009

45 (43)

34 (79%)

3 (7%)

6 (14%)

0

0

1 (2%)

1 (2%)

2010

41 (34)

26 (76%)

4 (12%)

3 (9%)

0

1 (3%)

5 (12%) 2 (5%)

2011

57 (51)

47 (92%)

1 (2%)

2 (4%)

0

1 (2%)

6 (11%) 0

Seroincident samplesd

2006 2007 2008 2009 2010 2011 Subtotal Trend p-value

All samplesd


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Total

489 (425)

Trend p-value a

349 (82%)

23 (5%)

43 (10%)

0.92

0.21

0.29

3 (1%) 0.12

7 (2%) 0.97

47 (10%) 17 (3%) 0.38

Number 1 of samples genotyped by MHA and number of samples which could be typed. Dual infections

were 2 classified separately from recombinants b

Number 3 and percentage (%) of given HIV-1 subtype (non-typeable and non-amplifiable samples

excluded 4 from denominator) c

NT=Non-typeable 5 samples, NA = Non-amplifiable samples presented by number and percentage (%)

where 6 number of samples genotyped was used as the denominator d

Seroprevalent 7 samples were defined as HIV-positive samples identified at screening; seroincident

samples 8 defined as HIV-positive samples identified after enrollment; all samples included both seroprevalent 9 and seroincident samples 10 11 12 13 14 15 16 17

0.17


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24

1 2

AIDS Research and Human Retroviruses Increasing HIV-1 Molecular Complexity among MSM in Bangkok (doi: 10.1089/AID.2014.0139) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. Page 25 of 26

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1 2


Page 26 of 26

26 1 2

Appendix Table 1 Subtyping of Seroincident and Seroprevalent Infections

3

Samples tested

Seroprevalent 278

Seroincident 211

Total 489

Typed by MHA

227/278 (82%)

158/211 (75%)

385 (79%)

4/278 (1%)

36/211 (17%)

40 (8%)

Amplifiable, nontypeable (NT)

34/278 (12%)

13/211 (6%)

47 (10%)

Non-amplifiable (NA)

13/278 (5%)

4/211 (2%)

17 (3%)

Typed by sequencing after MHAa

4 5 6 7 8 9

Increasing trends of syphilis among men who have sex with men in high income countries.

HIV Risk Behavior Among Men Who Have Sex with Men.

Increasing hepatitis A immunity in men who have sex with men in Sydney, 1996-2012.

Sex work among men who have sex with men and transgender women in Bogotá.

Engagement in group sex among geosocial networking mobile application-using men who have sex with men.

Predictors of HIV Sexual Risk Behavior among Men Who Have Sex with Men, Men Who Have Sex with Men and Women, and Transgender Women.

Intimate Partner Violence and Sex Among Young Men Who Have Sex With Men.

AIDS in Rio de Janeiro.

Longitudinal analysis of key HIV-risk behavior patterns and predictors in men who have sex with men, Bangkok, Thailand.

Outcomes associated with anxiety and depression among men who have sex with men in Estonia.

The Lisbon Cohort of men who have sex with men.

Factors Associated with Inconsistent Condom Use among Men Who Have Sex with Men in Cambodia.

HPV Prevalence in Multiple Anatomical Sites among Men Who Have Sex with Men in Peru.

Increase in Sexually Transmitted Infections among Men Who Have Sex with Men, England, 2014.

Culture, social networks and HIV vulnerability among men who have sex with men in Indonesia.

Intimate partner, familial and community violence among men who have sex with men in Namibia.

AIDS epidemic among men who have sex with men in China.

Risk factors for syphilis among married men who have sex with men in China.

Violence and depression among men who have sex with men in Tanzania.

Condom use and HIV testing among men who have sex with men in Jordan.

Condomless Vaginal Intercourse and Its Associates among Men Who Have Sex with Men in China.

Homonegativity and Associated Factors Among Men Who Have Sex with Men in Estonia.

Poppers use and risky sexual behaviors among men who have sex with men in Beijing, China.

HIV-1 transmission networks among men who have sex with men in Asia.