ORIGINAL ARTICLE
Risk group characteristics and viral transmission clusters in South-East Asian patients infected with human immunodeficiency virus-1 (HIV-1)
circulating recombinant form (CRF) 01_AE and subtype B
Rebecca A. Oyomopito
a, Yen-Ju Chen
b, Somnuek Sungkanuparph
c, Rami Kantor
d, Tuti Merati
e, Wing-Cheong Yam
f, Thira Sirisanthana
g, Patrick C.K. Li
f, Pacharee Kantipong
h, Praphan Phanuphak
i,
Chris K.C. Lee
j, Adeeba Kamarulzaman
k, Rossana Ditangco
l,
Szu-Wei Huang
b, Annette H. Sohn
m, Matthew Law
a, Yi Ming A. Chen
b,n,*
aThe Kirby Institute, University of New South Wales, Sydney, Australia
bCenter for Infectious Disease and Cancer Research, Kaohsiung Medical University, Kaohsiung City, Taiwan
cFaculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand
dDivision of Infectious Diseases, Brown University Alpert Medical School, RI, USA
eFaculty of Medicine Udayana University & Sanglah Hospital, Bali, Indonesia
fDepartment of Microbiology, Queen Mary Hospital, Faculty of Medicine, The University of Hong Kong, Hong Kong, China
gResearch Institute for Health Sciences, Chiang Mai University, Chiang Mai, Thailand
hChiangrai Prachanukroh Hospital, Chiangrai, Thailand
iHIV-NAT/Thai Red Cross AIDS Research Centre, Bangkok, Thailand
jHospital Sungai Buloh, Sungai Buloh, Malaysia
kUniversity Malaya Medical Centre, Kuala Lumpur, Malaysia
lResearch Institute for Tropical Medicine, Manila, Philippines
mTREAT Asia, amfARdThe Foundation for AIDS Research, Bangkok, Thailand
nDepartment of Microbiology, Kaohsiung Medical University, Kaohsiung City, Taiwan
Received 17 December 2014; accepted 25 June 2015 Available online 21 August 2015
Conflicts of interest: All authors declare no conflicts of interest.
* Corresponding author. Department of Microbiology and Center for Infectious Disease and Cancer Research, Kaohsiung Medical University, 100 Shiih-Chuan 1st Road, Kaohsiung City, 80708, Taiwan.
E-mail address:[email protected](Y.M.A. Chen).
http://dx.doi.org/10.1016/j.kjms.2015.07.002
1607-551X/Copyrightª2015, Kaohsiung Medical University. Published by Elsevier Taiwan LLC. All rights reserved.
Available online atwww.sciencedirect.com
ScienceDirect
journal home page:http:/ /www.kjms-onli ne.com
KEYWORDS Asia;
HIV exposure;
CRF01_AE;
Subtype B
Abstract Human immunodeficiency virus (HIV)-1 epidemics in Asian countries are driven by varying exposures. The epidemiology of the regional pandemic has been changing with the spread of HIV-1 to lower-risk populations through sexual transmission. Common HIV-1 genotypes include subtype B and circulating recombinant form (CRF) 01_AE. Our objective was to use HIV-1 genotypic data to better quantify local epidemics. TASER-M is a multicenter prospective cohort of HIV-infected patients. Associations between HIV exposure, patient sex, country of sample origin and HIV-1 genotype were evaluated by multivariate logistic regression. Phylogenetic methods were used on genotypic data to investigate transmission relationships. A total of 1086 patients from Thailand, Hong Kong, Malaysia and the Philippines were included in analyses.
Proportions of male patients within countries varied (Thailand: 55.6%, Hong Kong: 86.1%, Malaysia: 81.4%, Philippines: 93.8%;p<0.001) as did HIV exposures (heterosexual contact:
Thailand: 85.7%, Hong Kong, 46.2%, Malaysia: 47.8%, Philippines: 25.0%;p<0.001). After adjust- ment, we found increased subtype B infection among men who have sex with men, relative to heterosexual-reported exposures (odds ratioZ2.4,p<0.001). We further describe four trans- mission clusters of eight to 15 treatment naı¨ve, predominantly symptomatic patients (two each for subtype B and CRF01_AE). Risk-group subpopulations differed with respect to the infecting HIV-1 genotype. Homosexual exposure patients had higher odds of being infected with subtype B. Where HIV-1 genotypes circulate within countries or patient risk-groups, local monitoring of genotype-specific transmissions may play a role in focusing public health prevention strategies.
Phylogenetic evaluations provide complementary information for surveillance and monitoring of viruses with high mutation rates such as HIV-1 and Ebola.
Copyrightª2015, Kaohsiung Medical University. Published by Elsevier Taiwan LLC. All rights reserved.
Introduction
Human immunodeficiency virus (HIV)-1 infections are dominated by group M viruses which are classified into nine different subtypes (AeD, FeH, J, K). Circulating recombi- nant forms (CRFs) result from recombination between HIV-1 genotypes (subtypes or CRFs) within a dually infected person [1]. The AsiaePacific, representing more than 60% of the world’s population is second only to sub-Saharan Africa in HIV-infection prevalence (5 million) and incidence (14%)[2].
In Asia, country-specific epidemics feature different HIV genotypes and epidemics are of increasing viral diversity.
Common regional genotypes are subtypes B and C, CRF01_AE and their recombinants. Cambodia, Myanmar, Thailand and Vietnam have been historically dominated by CRF01_AE, in China’s Special Administrative Region of Hong Kong subtype B and CRF01_AE cocirculate, mainland China infections include B/C recombinants and subtype B in- fections are common in Japan[3]. Previously, we reported on Asian patients who were predominantly infected with CRF01_AE and subtype B[4]. Among patients for whom both HIV-1 protease and reverse transcriptase genotypes were determined, 6.4% were infected with discordant protease and reverse transcriptase genotypes, consisting mainly of subtype B and CRF01_AE components.
HIV genotype diversity is associated with the heteroge- neity in the human subpopulations driving regional Asian epidemics [5]. Sexual transmission accounts for most in- fections globally. However, in the AsiaePacific, epidemics are diverse with concentrated, generalized and low- prevalence epidemics predominating in different coun- tries. In many countries, epidemics have been historically
concentrated in high-risk groups such as people who inject drugs (including street children), men who have sex with men (MSM), sex workers, their clients, and sexual partners.
The epidemiology of the Asian pandemic is changing with HIV-1 being increasingly spread to lower-risk populations with onward sexual transmission to female partners of high- risk groups helping to sustain generalized epidemics[5].
Phylogenetic analysis is a method of reconstructing re- lationships between genotypic sequences and is one strat- egy for studying viral transmission dynamics in humans.
Phylogenetic studies cannot determine the direction of HIV-1 evolution or, consequently, the direction of trans- mission [6]. Classification of like sequences into clusters can, however, identify transmission networks thereby helping to quantify the evolving roles of cocirculating ge- notypes[7]. Although transmission network evaluations are not routinely incorporated in incidence reporting.
Under the TREAT Asia Studies to Evaluate Resistance monitoring (TASER-M) protocol [8], patient characteristics are collected and infecting HIV-1 isolates are genotypically sequenced. We used TASER-M baseline data to evaluate associations between patient risk group and infecting ge- notype. For additional context, we complemented findings with descriptions of transmission clusters among our patients.
Methods
Patients were enrolled in a multicenter, prospective cohort [8]. Covariates included age at study entry, sex, patient- reported HIV exposure, hepatitis B (HBV) and hepatitis C (HCV) coinfections, and indices of illness severity [CD4 T
lymphocyte count, HIV-1 RNA and Centers for Disease Control and Prevention (CDC) classification][9]. The most severe pre-TASER-M CDC category was used as baseline clinical status. HBV (HCV) positive status was defined as having any HBsAg (HCV-Ab) positive result prior to enrol- ment. Patients reporting dual HIV-exposure routes were classified under the category of higher risk. For individual country analyses, due to small numbers of patients reporting injecting drug use (IDU), infection by blood products or having unknown exposure, these modes of infection were excluded.
For univariate comparisons by country,c2, Fisher’s exact or KruskaleWallis tests were performed, as appropriate.
Associations between patient-reported HIV-exposure, sex, country of isolate origin and infecting HIV-1 genotype (subtype B or CRF01_AE) were evaluated by multivariate logistic regression, for individual countries and overall.
Forward step-wise techniques were used to determine best fitting models. Binary covariate and multicategorical parameterpvalues<0.2 (tests for trend/heterogeneity), in univariate assessments, were considered for multivariate models. Final multivariate models consisted of covariates remaining significant atp<0.05. Analyses were performed using SAS version 9.2 (SAS Institute Inc., Cary, NC, USA).
Phylogenetic analysis was used to identify transmission clusters among patients. Sequences not passing quality control evaluations (Virco BVBA, Beerse, Belgium) or with missing bases or gaps were excluded. By genotype, remaining sequences were trimmed using Clustal W, implemented in MEGA version 5 [10]. HXB2 and two CRF01_AE reference strains (TH.90, TH93,http://www.hiv.
lanl.gov) were used to align subtype B and CRF01_AE se- quences, respectively. Each reference strain set (B, CRF01_AE) served as outgroup for the other.
Phylogenetic topologies were generated by HIV Sequence Database PhyML (v2.4.4) using the method of maximum likelihood and general time reversible (GTR) plus gamma nucleotide substitution model, verified on the data [11]. Subtype B sequences were analyzed as a group. Due to resource limitations imposed by computations for the larger number of CRF01_AE sequences, phylogenies were gener- ated by country. For Thailand, sequences were further separated into Bangkok metropolitan (Thai Metro) or regional urban centers (Thai Urban: Chiang Mai, Chiang Rai). Clustering was determined using bootstrapping with an acceptable 100 replicates.
Nucleotide sequence accession numbers
The HIV-1 sequences reported in this study have been deposited in GenBank with accession numbers KC791222 to KC791224, KC791226 to KC791228, KC791230 to KC791236, KC791238 to KC791269, KC791271 to KC791285, KC791288 to KC791297, KC791299 to KC791314, KC791316 to KC791344, KC791346 to KC791354, KC791356 to KC791363, KC791365, KC791366, KC810320 to KC810322, KC810324, KC810326 to KC810329, KC810331 to KC810386, KC810388 to KC810396, KC810398 to KC810410, KC810412 to KC810424, KC810426 to KC810431, KC810433 to KC810487, KC810489 to KC810501, KC810503 to KC810510, KC810512 to KC810517, KC810544 to KC810548, KC810550 to
KC810557, KC810559 to KC810562, KC810564, KC810565, KC810567 to KC810573, KC810575, KC810577 to KC810580, KC810582 to KC810637, KC810639 to KC810647, KC810649 to KC810661, KC810663 to KC810675, KC810677 to KC810682, KC810684 to KC810738, KC810740 to KC810752, KC810754 to KC810761, KC810763 to KC810768, KC810795 to KC810799, KC810801 to KC810808, KC810810 to KC810813, KC810815, KC810816, KC810818 to KC810821, KC856945, KC856946, KC856947 to KC856950, KC856952 to KC856960, to KC856980, KC856982, KC856983, KC856985 to KC857009, KC857011 to KC857021, KC857023 to KC857029, KC857031 to KC857042, KC857044 to KC857052, KC857054 to KC857056, KC857058 to KC857061, KC857063 to KC857084, KC857105, KC857107 to KC857111, KC857113 to KC857121, KC857123 to KC857141, KC857143, KC857144, KC857146 to KC857170, KC857172 to KC857182, KC857184 to KC857190, KC857192 to KC857203, KC857205 to KC857213, KC857215 to KC857217, KC857219 to KC857222, KC857224 to KC857245, KC867560 to KC867616, KC867618 to KC867620, KC867622 to KC867647, KC921421, KC921422, KC921767, KC921769 to KC921774, KC921776 to KC921783, KC921787, KC921789 to KC921793, KC921795 to KC921797, KC921799 to KC921805, KC921807 to KC921822, KC921827 to KC921829, KC921832, KC921834, KC921836 to KC921842, KC921844 to KC921848, KC921850, KC921852 to KC921859, KC921861 to KC921866, KC921868 to KC921872, KC921874 to KC921880, KC921882 to KC921887, KC921889 to KC921896, KC921900, KC921902 to KC921906, KC921908 to KC921910, KC921912 to KC921918, KC921920 to KC921935, KC921940 to KC921942, KC921945, KC921947, KC921949 to KC921955, KC921957 to KC921961, KC921963, KC921965 to KC921972, KC921974 to KC921979, KC921981 to KC921985, KC921987 to KC921992, KC970855 to KC970858, KC970861, KC970862, KC970865, KC970869 to KC970871, KC970874, KC970875, KC970878, KC970882, KC970890, KC970894, KC970907, KC970909, KC970911, KC970914, KC970915, KC970924 to KC970928, KC970931, KC970933, KC970936, KC970938, KC970945 to KC970947, KC970949, KC970952, KC970954, KC970958, KC970961, KC970964, KC970966, KC970967, KC970970, KC970971, KC970976, KC970978, KC970979, KC970982 to KC970985, KC970988, KC970989, KC970992, KC970996, KC970998, KC970999, KC971002, KC971003, KC971006 to KC971009, KC971012 to KC971014, , KC971023, KC971025, KC971028, KC971030, KC971033 to KC971035, KC971041, KC994162 to KC994169, KC994171 to KC994182, KC994184 to KC994186, KC994188 to KC994190, KC994193 to KC994196, KC994198 to KC994202, KC994204 to KC994211, KC994213 to KC994220, KC994223 to KC994225, KC994227 to KC994234, KC994236 to KC994241, KC994243, KC994245, KC994247 to KC994249, KC994251 to KC994260, KC994262 to KC994264, KC994267, KC994269, KC994271 to KC994273, KC994275 to KC994279, KC994281, KC994342 to KC994360, KC994362, KC994364 to KC994368, KC994371 to KC994376, KC994379, KC994380, KC994383 to KC994387, KC994451, KC994452, KF273871 to KF273901.
Results
A total of 1086 patients enrolled between 2007 and 2010 from nine clinic locations in Thailand (nZ4), Hong Kong (China;
nZ2) Malaysia (nZ2), and the Philippines (nZ1) were
included. Excepting antiretroviral therapy experience, dif- ferences existed for all covariates shown inTable 1. Hong Kong patients were older (p< 0.001) and proportions of males within countries varied (p< 0.001). Patients from Thailand more frequently reported exposure as heterosexual contact (p<0.001). Testing differed between countries for HBV (p<0.001) and HCV (p<0.001). In the 95% of patients naı¨ve to antiretroviral therapy, country differences were
found in CD4 counts (p<0.001), log10HIV-1 RNA copies/mL (p<0.001) and CDC classification (p<0.001).
Of all genotypes, approximately 98% of patients were infected with either CRF01_AE [nZ882 (81.2%)] or subtype B [nZ178 (16.4%)]. Due to the small numbers of patients from some countries, HIV exposure was restricted to het- erosexual and homosexual contact in country-specific ana- lyses. Adjusted estimates for Hong Kong patients,
Table 1 Patient characteristics by country.
Thailand Hong Kong Malaysia Philippines Total
nZ768 (70.7%) nZ173 (15.9%) nZ113 (10.4%) nZ32 (2.9%) nZ1086 (99.9%) Age (y),
Median (IQR) 36.7 (31.2e42.5) 41.3 (33.9e49.7) 35.7 (31.1e44.7) 34.3 (29e41.2) 36.8 (31.6e44.4) Sex
Male 427 (55.6) 149 (86.1) 92 (81.4) 30 (93.8) 698 (64.3)
Female 341 (44.4) 24 (13.9) 21 (18.6) 388 35.7)
Ethnicity
Caucasian 0 (0.0) 5 (2.9) 1 (0.9) 0 (0.0) 6 (0.6)
Chinese 0 (0.0) 152 (87.9) 51 (45.1) 0 (0.0) 203 (18.7)
Indian 0 (0.0) 0 (0.0) 14 (12.4) 0 (0.0) 14 (1.3)
Malay 0 (0.0) 0 (0.0) 44 (38.9) 0 (0.0) 44 (4.1)
Filipino 0 (0.0) 1 (0.6) 0 (0.0) 32 (100.0) 33 (3.0)
Thai 761 (99.1) 6 (3.5) 0 (0.0) 0 (0.0) 767 (70.6)
Other 7 (0.9) 9 (5.2) 3 (2.7) 0 (0.0) 19 (1.7)
HIV exposure
Homosexual contact 84 (10.9) 86 (49.7) 26 (23.0) 24 (75.0) 220 (20.3)
Homosexual & IDU 1 (0.1) 0 (0.0) 1 (0.9) 0 (0.0) 2 (0.2)
IDU only 4 (0.5) 1 (0.6) 16 (14.2) 0 (0.0) 21 (1.9)
Heterosexual contact 658 (85.7) 80 (46.2) 54 (47.8) 8 (25.0) 800 (73.7)
Heterosexual & IDU 11 (1.4) 1 (0.6) 4 (3.5) 0 (0.0) 16 (1.5)
Receipt of blood/products 2 (0.3) 1 (0.6) 0 (0.0) 0 (0.0) 3 (0.3)
Perinatal transmission 1 (0.1) 0 (0.0) 0 (0.0) 0 (0.0) 1 (0.1)
Unknown 7 (0.9) 4 (2.3) 12 (10.6) 0 (0.0) 23 (2.1)
Hepatitis B
Negative 452 (58.9) 144 (83.2) 93 (82.3) 13 (40.6) 702 (64.6)
Positive 54 (7.0) 17 (9.8) 5 (4.4) 2 (6.3) 78 (7.2)
Not tested 262 (34.1) 12 (6.9) 15 (13.3) 17 (53.1) 306 (28.2)
Hepatitis C
Negative 401 (52.2) 143 (82.7) 75 (66.4) 0 (0.0) 619 (57.0)
Positive 33 (4.3) 8 (4.6) 20 (17.7) 0 (0.0) 61 (5.6)
Not tested 334 (43.5) 22 (12.7) 18 (15.9) 32 (100.0) 406 (37.4)
ARV experience
Naı¨ve 722 (94.0) 168 (97.1) 113 (100.0) 30 (93.8) 1033 (95.1)
First-line experienced 36 (4.7) 5 (2.9) 0 (0.0) 2 (6.3) 43 (4.0)
ARVs for MTCT 10 (1.3) 0 (0.0) 0 (0.0) 0 (0.0) 10 (0.9)
Baseline CD4 count (cells/mL)a
Median (IQR) 96 (35e195) 61 (19.5e173.5) 153.5 (40e226) 227 (143e303) 97 (32e200) Log10HIV-1 viral load (copies/mL)a
Median (IQR) 5 (4.6e5.3) 5.2 (4.9e5.6) 5.1 (4.6e5.4) 5.2 (5e5.6) 5 (4.6e5.4) CDC classificationa
Category A 315 (43.6) 63 (37.5) 96 (85.0) 18 (60.0) 492 (47.6)
Category B 113 (15.7) 21 (12.5) 0 (0.0) 2 (6.7) 136 (13.2)
Category C 294 (40.7) 84 (50.0) 17 (15.0) 10 (33.3) 405 (39.2)
Data are presented asn(%), unless otherwise indicated.
ARVZantiretroviral; CDCZCenters for Disease Control and Prevention; HIVZhuman immunodeficiency virus; IDUZinjecting drug use; IQRZinterquartile range; MTCTZmother-to-child transmission.
a Country results for CDC class, baseline CD4 counts and HIV-1 RNA are for ARV-naı¨ve patients only. There were 18 (24) patients missing HIV-1 viral load (CD4 counts) at baseline.
suggested that homosexual contact had higher odds of subtype B infection [odds ratio (OR) Z 3.1; p < 0.001]
whereas women had lower odds (ORZ0.164;p<0.023). In Thailand patients, after adjustment for HIV exposure, women had lower odds of subtype B infection (ORZ0.396;
p < 0.008). In Malaysia, no associations were found, possibly due to small cell sizes. There were insufficient data from the Philippines to perform the analysis.
For logistic regression analyses including all countries, patients having any IDU exposure were collapsed into one category,Any IDU. Univariate and adjusted estimates for all countries are shown inTable 2. After adjustment, females had lower odds of subtype B infection (OR Z 0.4, p<0.003). The overall heterogeneity testpvalue must be significant before category effects can be interpreted as contributing and both HIV-exposure (p<0.001) and country (p< 0.001) were associated with infecting genotype. Pa- tients reporting homosexual exposure had higher odds of being infected with subtype B, compared with heterosexual exposure (ORZ2.4,p< 0.001). Compared with patients from Thailand, patients from Hong Kong (OR Z 8.9, p < 0.001), Malaysia (OR Z 3.6, p < 0.001), and the Philippines (OR Z 10.4, p < 0.001) had higher odds of subtype B infection.
Following quality control and sequence preparation, there were 114 subtype B and 753 CRF01_AE sequences from Thailand, Hong Kong, and Malaysia remaining for phylogenetic reconstruction. Subtype B analysis was per- formed on all sequences of this subtype, resulting in 15 groups [bootstrap value (BV)>70%] comprising of clusters of 15 and nine, two triplets and 11 pairs (Figure 1). The cluster of 15 sequences (BVZ99%) included men attending two sites in Hong Kongdone patient reporting heterosexual exposure, the others self-identifying as MSM [age in years:
median: 40.7, minimum: 20.8, maximum: 58.6;
symptomatic (CDC C or B):nZ 11/15 (73.3%)]. The nine- group cluster (BV Z 99%) were male patients from two sites in Bangkok comprising of six patients reporting het- erosexual contact and three MSM exposures [age in years:
median: 30.8, minimum: 26.5, maximum: 36.0; symptom- atic:nZ5/9 (55.6%)]. Malaysian clusters included a male heterosexual/MSM pair, one each from two sites in Kuala Lumpur (BV Z 100%). The only clustering female isolate was similar to that of a male patient reporting unknown exposure.
Due to the large number of sequences, CRF01_AE ana- lyses were performed individually for Hong Kong, Malaysia, Thai Metro, and Thai Urban. Ten groups were detected among 70 patients from Hong Kong (Figure 2); a cluster of 10 [heterosexual; male:nZ8, female:nZ2; age in years:
median: 55.7, minimum: 34.2, maximum: 70.8; symptom- atic:nZ8/10 (80.0%), BVZ98%)], a cluster of eight [MSM:
nZ6, heterosexual female:nZ1, male IDU:nZ1; age in years: median: 41.0, minimum: 26.0, maximum: 46.7;
symptomatic:nZ5/8 (62.5%), BVZ100%], and eight pairs (BVs>70%). There was a relatively small amount of clus- tering found in collections of Thai Metro (nZ 288; pairs:
nZ4, BVs99%) or Thai Urban sequences (nZ344; pairs:
nZ9, triplets:nZ1; BVs70%). For the 51 isolates from Malaysia (Figure 3), five clusters were found; two clusters of four (BV>91%), one including patients from two sites in Kuala Lumpur, and one pair (BV Z 79%). A subset of 14 CRF01_AE sequences clustered closer to outgroup subtype B and were verified using HIV Sequence Database BLAST (http://www.hiv.lanl.gov). Of these, one pair and one triplet included three IDU males (BV>86%).
Overall for subtype B or CRF01_AE, patients in groups of eight or more (nZ42) were treatment-naı¨ve, aged 21e71 years [median: 39.8 years, interquartile range (IQR):
31.8e47.3], symptomatic [n Z 29/42 (69.1%)], with low
Table 2 Factors associated with subtype B infection
n No. Univariate analysis Multivariate analysis
Subtype B n(%)
OR p OR (95% CI) p
Patient covariates Sex
Male 681 159 (23.3)
Female 379 19 (5.0) 0.17 <0.001 0.43 (0.2e0.7) 0.003
HIV exposure
Heterosexual contact 781 74 (9.5) <0.001 <0.001
Homosexual contact 216 92 (42.6) 7.09 <0.001 2.37 (1.5e3.7) <0.001
Any IDU exposure 39 5 (12.8) 1.41 0.492 0.74 (0.3e2.1) 0.566
Unknown 24 7 (29.2) 3.93 0.003 2.21 (0.8e6.1) 0.127
Country
Thailand 763 51 (6.7) <0.001 <0.001
Hong Kong, China 161 82 (50.9) 14.49 <0.001 8.89 (5.7e13.9) <0.001
Malaysia 109 28 (25.7) 4.83 <0.001 3.63 (2.1e6.4) <0.001
Philippines 27 17 (63.0) 23.73 <0.001 10.42 (4.3e25.2) <0.001
Total 1060 178 (16.8)
Reference categories for sex, HIV exposure, and country are male, heterosexual contact and Thailand respectively;pvalues for HIV exposure and country categories are test for homogeneity.
CIZconfidence interval; HIVZhuman immunodeficiency virus; IDUZinjecting drug use; ORZodds ratio.
X120 44 R731 75 R721 77 R731 91 R730 02 R721 83
R410 65 Ref.B.FR.8
X120 31 X120 08
X120 03 R720 44
R720 33 R120 19 R120 12
R420 28 R120 47 R710 60 R120 31
R410 40 R121 42
R120 83 X120 53 X120 39
R120 99 X120 11
R720 92 X120 48 R120 69 R120 64
R121 43 R120 15
R121 13 R120 71 R120 57 R120 56
R121 40 R120 93 R120 95 R120 53 R120 14
R121 04 R420 72 X120 16
R120 72 R120 35 X120 14
R720 14 R710 48
R120 24 R731 72
R121 23 R120 82
R121 15 R120 50 R120 37 X120 21
R121 07 R120 77 R721 74
R120 51 R722 44 R710 04 R722 00 R721 29
R710 19 R711 91
R721 10 R710 70 R721 39 R120 02
R721 59 R120 68
R420 76 R120 13
R120 58 R120 09 R120 03
R720 04 R120 84 R120 79 R120 36
X120 51 R120 73
R120 43 R411 08 R121 31 R121 16
X120 20 X120 04 R121 24
R711 08 R410 19
R121 39 R120 49
R120 63 R722 37 R721 41
R711 95 R420 37 R720 74 R720 30
R721 57
R420 57 R410 59
X120 41 R121 34 R121 17
X120 18 X120 02
R120 98
R721 07 R121 14
R731 25 R120 78
Ref01 AE1.
Ref01 AE2.
21 10
94 11
56 1
35 20
100 49
17 1 0
24 20 1
22
22 2
19
9778
32 92
80 70 93 66
99
40
16 1 0
42 17 9
98 100 26
3 0 0
100 83
14 2
62
73
49 63 58 50
99 7
19 4423
100 55
0
56
96 13
0 0
100 55
100 14
22 100 1
64 100 18
0
25
51 23 55 30
100 14
92 12 6
43 21
46
52
97
100
0.02
Hong Kong
Bangkok
X120 48 R120 69 R120 64
R121 43 R120 15
R121 13 R120 71 R120 57 R120 56
R121 40 R120 93 R120 95 R120 53 R120 14
R121 04 9778
3332 92
80 70 93
6 Hong Kong
R722 44 R710 04 R722 00 R721 29
R710 19 R711 91
R721 10 R710 70 R721 39 62
73
49 63 58 50
Bangkok
Figure 1. Phylogenetic reconstructiondsubtype B sequences. Branches in red leading to groups of anonymous patient identifiers show those groups of sequences with bootstrap statistics>80%. Clusters of 15 (Hong Kong, China) and nine (Bangkok, Thailand) sequences are boxed in blue.
CD4 counts (median: 80 cells, IQR: 29e180), and high viral load (median: 177,901 copies/mL, IQR: 84,000e406,100).
Discussion
This large-scale study of HIV-1 genotypes in patients from South-East Asia showed country-specific differences in HIV epidemiology and increased subtype B infection among MSM
patients, relative to patients reporting heterosexual expo- sures. Overall, one half of treatment-naı¨ve patients were asymptomatic. By contrast, treatment-naı¨ve patients in clusters of eight or more, were predominantly symptomatic with enhanced capacity for onward transmission.
Our study limitations include that TASER-M was not specifically designed to provide regional surveillance esti- mates. Intermediary transmission events outside our cohort are also unknown. Consequently, patients and networks
X12022 R12017 R12101
R12135
X12033 R12129 R12070
R12021 R12119
X12024
R12138 R12106
R12133 X12007
X12043 R12005
X12038 R12041
R12032
R12111 X12052
R12120
R12144 R12033
R12028 X12047
R12112 R12081
X12032 R12016 R12001
R12067 R12145 X12017 Ref1.01AE
R12023 R12122
R12118 Ref2.01AE
X12023 R12087
R12010 R12042
R12137 R12006 R12004 X12030
X12005 R12060
R12059 R12088
X12042 R12061
X12009 R12147 R12066
R12022 R12018 X12055
X12054 X12027
X12012 R12094 R12097
R12108 R12086 X12029
X12001 R12091
R12052 R12007
R12074
Ref.B.FR.8
99
81
74
89 98
95 10 0
72
10 0 98
10 0
99
10 0
0.00 5
R12033 R12028
X12047 R12112 R12081
X12032 R12016 R12001
74
89 98
R12042
R12137 R12006 R12004 X12030
X12005 R12060
R12059 R12088
X12042
95 10 0
Figure 2. CRF01_AE phylogenetic reconstructiondHong Kong. Branches in red leading to groups of anonymous patient identifiers show those groups of sequences with bootstrap statistics>80%. Clusters of 15 and eight sequences are boxed in blue.
cannot be considered entirely representative of countries or the region. Computing restrictions possibly impacted the precision of our phylogenetic estimates, however, the methods are sufficiently robust to support interpretations.
Additional investigation of Malaysian sequences, including those for people who inject drugs, is required. Malaysia is an environment where extensive recombination is reported and sequence variation may have reflected this [12].
Sequence preparation may have artifactually impacted on interpretations. Nevertheless, with the large number of contributing sequences, misclassification effects would be small.
In Asia, drivers of country-level HIV-1 epidemics are diverse[5]and our results show that different viral geno- types can predominate in specific patient risk-groups. Local prevention strategies need to target subpopulations at-risk for regional reductions. HIV-1 genotype is routinely avail- able from drug resistance testing. Therefore, infecting HIV-1 genetic information from phylogenetic studies, part- nered with assessment of de-identified patient character- istics, could enhance local epidemic surveillance and
monitoring to better focus prevention efforts. Timely reporting of changes in HIV-1 genotype distributions, including entry of novel variants into local settings, could inform public health strategies and help arrest proliferation of HIV-1 clusters into transmission networks. Phylogenetic assessments may be used to enhance tracking of HIV-1 and other disease entities with high mutation rates, such as Ebola[13].
Sources of funding
The TREAT Asia HIV Observational Database, TREAT Asia Studies to Evaluate Resistance, and the Australian HIV Observational Database are initiatives of TREAT Asia, a program of amfAR, The Foundation for AIDS Research, with support from the Dutch Ministry of Foreign Affairs through a partnership with Stichting Aids Fonds, and the U.S. National Institutes of Health’s National Institute of Allergy and In- fectious Diseases, Eunice Kennedy Shriver National Institute of Child Health and Human Development, and National
R4206 0 R4108 5
R4103 6 R41052 R4100 4 R4106 6
R4113 1 R4101 8 R4114 9
R4103 5 R4108 1 R4104 4 R4111 2 R4202 2
R4115 0 R4111 0
R4106 2 R4205 6
R4112 0 R4103 7 Ref1.01 AE
Ref2.01 AE R4200 2
R4106 8 R4205 3
R4109 1 R4114 2 R4113 3 R4100 8
R4207 5 R4114 5 R4110 5
R4107 6 R4102 7
R4106 3 R4109 4 R4105 0 R4111 9 R4106 4
R4204 4 R4112 7 R4110 0 R4107 8 R4201 7 R4101 2
R4100 6 R4115 6
R4103 9 R4209 6 R4104 7 R4104 6
R4202 0 R4101 1 Ref.B.FR.8
100 91
95 98
79
100
94
86
100
0.02
R4206 0 R4108 5
R410300 6 R41052 100 91
R4114 9 R410300 5
R4108 1 R410400 4 95 99 98
R4112 0 R410300 7 79
77
R4110 0 R4107 8 94
99
R4101 2 R410000 6 R4115 6 86
Figure 3. CRF01_AE phylogenetic reconstructiondMalaysia. Branches in red leading to groups of anonymous patient identifiers show those groups of sequences with bootstrap statistics>80%. Clusters are boxed in blue.
Cancer Institute, as part of the International Epidemiologic Databases to Evaluate AIDS (IeDEA; U01AI069907). Queen Elizabeth Hospital and the Integrated Treatment Centre received additional support from the Hong Kong Council for AIDS Trust Fund. The Kirby Institute is funded by the Australian Government Department of Health and Ageing, and is affiliated with the Faculty of Medicine, University of New South Wales. The content of this publication is solely the responsibility of the authors and does not necessarily represent the official views of any of the institutions mentioned above.
Acknowledgments
A. Kamarulzaman,1 A. Kajindran, and L.Y. Ong, University Malaya Medical Center, Kuala Lumpur, Malaysia; C.K.C. Lee, R. David, and B.L.H. Sim, Hospital Sungai Buloh, Kuala Lumpur, Malaysia; C.V. Mean, V. Saphonn, and K. Vohith, National Center for HIV/AIDS, Dermatology and STDs, Phnom Penh, Cambodia; E. Yunihastuti, Working Group on AIDS, Faculty of Medicine, University of Indonesia/Cipto- mangunkusumo Hospital, Jakarta, Indonesia; F.J. Zhang, H.X. Zhao, and N. Han, Beijing Ditan Hospital, Capital Medical University, Beijing, China; J.Y. Choi, S.H. Han, and J.M. Kim, Division of Infectious Diseases, Department of Internal Medicine, Yonsei University College of Medicine, Seoul, South Korea; M. Mustafa and N. Nordin, Hospital Raja Perempuan Zainab II, Kota Bharu, Malaysia; N. Kumar- asamy, S. Saghayam, and C. Ezhilarasi, YRG Centre for AIDS Research and Education, Chennai, India; O.T. Ng, A. Chua, L.S. Lee, and A. Loh, Tan Tock Seng Hospital, Singapore;
P.C.K. Li1and M.P. Lee, Queen Elizabeth Hospital and K.H.
Wong, Integrated Treatment Centre, Hong Kong, China; P.
Kantipong and P. Kambua, Chiang Rai Prachanukroh Hospi- tal, Chiang Rai, Thailand; P. Phanuphak, K. Ruxrungtham, M. Khongphattanayothin, and S. Sirivichayakul, HIV-NAT/
Thai Red Cross AIDS Research Centre, Bangkok, Thailand;
R. Ditangco, E. Uy, and R. Bantique, Research Institute for Tropical Medicine, Manila, Philippines; R. Kantor, Brown University, RI, U.S.A.; S. Oka, J. Tanuma, and T. Nishijima, National Center for Global Health and Medicine, Tokyo, Japan; S. Pujari, K. Joshi, and A. Makane, Institute of In- fectious Diseases, Pune, India; S. Sungkanuparph, S. Kier- tiburanakul,2 L. Chumla, and N. Sanmeema, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand; T.P. Merati, D.N. Wirawan, and F. Yuli- ana, Faculty of Medicine, Udayana University and Sanglah Hospital, Bali, Indonesia; T. Sirisanthana, R. Chaiwarith, W.
Kotarathititum, and J. Praparattanapan, Research Institute for Health Sciences, Chiang Mai University, Chiang Mai, Thailand; T.T. Pham, D.D. Cuong, and H.L. Ha, Bach Mai Hospital, Hanoi, Vietnam; V.K. Nguyen, V.H. Bui, and T.T.
Cao, National Hospital for Tropical Diseases, Hanoi, Viet- nam; W. Ratanasuwan and R. Sriondee, Faculty of Medi- cine, Siriraj Hospital, Mahidol University, Bangkok, Thailand; Y.M.A. Chen, W.W. Wong, Y.J. Chen, L.H. Kuo,
S.W. Huang and Y.T. Lin, Taipei Veterans General Hospital and AIDS Prevention and Research Centre, National Yang- Ming University, Taipei, Taiwan; A.H. Sohn, N. Durier, B.
Petersen, and T. Singtoroj, TREAT Asia, amfAR - The Foundation for AIDS Research, Bangkok, Thailand; D.A.
Cooper, M.G. Law, and A. Jiamsakul, The Kirby Institute, University of New South Wales, Sydney, Australia.
References
[1]Korber B, Gaschen B, Yusim K, Thakallapally R, Kesmir C, Detours V. Evolutionary and immunological implications of contemporary HIV-1 variation. Br Med Bull 2001:19e42.
[2]WHO. Global report: UNAIDS report on the global AIDS epidemic. 2010.
[3]Hemelaar J, Gouws E, Ghys PD, Osmanov S. Global and regional distribution of HIV-1 genetic subtypes and recombi- nants in 2004. AIDS (London, England) 2006;20:W13e23.
[4]Oyomopito RA, Li PC, Sungkanuparph S, Phanuphak P, Tee KK, Sirisanthana T. Evaluating immunologic response and clinical deterioration in treatment-naive patients initiating first-line therapies infected with HIV-1 CRF01_AE and subtype B. J Acquir Immune Defic Syndr 2013;62:293e300.
[5]Oyomopito R. The HIV-1 group M epidemic in the Asia-Pacific.
In: Murray JE, editor. Exposure therapy: new developments.
Hauppauge: Nova Science Publishers, Inc; 2012. p. 287e99.
Available at:.
[6]Kaye M, Chibo D, Birch C. Comparison of Bayesian and maximum-likelihood phylogenetic approaches in two legal cases involving accusations of transmission of HIV. AIDS Res Hum Retroviruses 2009;25:741e8.
[7]Takebe Y, Liao H, Hase S, Uenishi R, Li Y, Li XJ, et al.
Reconstructing the epidemic history of HIV-1 circulating re- combinant forms CRF07_BC and CRF08_BC in East Asia: the relevance of genetic diversity and phylodynamics for vaccine strategies. Vaccine 2010;28(Suppl. 2):B39e44.
[8]Hamers RL, Oyomopito R, Kityo C, Phanuphak P, Siwale M, Sungkanuparph S. Cohort profile: the PharmAccess African (PASER-M) and the TREAT Asia (TASER-M) monitoring studies to evaluate resistancedHIV drug resistance in sub-Saharan Af- rica and the Asia-Pacific. Int J Epidemiol 2012;41:43e54.
[9]Centers for Disease Control and Prevention (CDC). Revised surveillance case definitions for HIV infection among adults, adolescents, and children aged<18 months and for HIV infec- tion and AIDS among children aged 18 months to<13 yearsd- United States, 2008. MMWR Recomm Rep 2008;57:1e16.
[10] Tamura K, Peterson D, Peterson N, Stecher G, Nei M. MEGA5:
molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 2011;28:2731.
[11] Guindon S. A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol 2003;52:
696e704.
[12] Lau KA, Wang B, Kamarulzaman A, Ngb KP, Saksena NK.
Continuous crossover(s) events of HIV-1 CRF01_AE and B sub- type strains in Malaysia: evidence of rapid and extensive HIV-1 evolution in the region. Curr HIV Res 2008;6:108e16.
[13] Thurmond J, Dimitrijevic M, Yoon H. The LANL hemorrhagic fever virus database, a new platform for analyzing biothreat viruses. Nucleic Acids Res 2012;40:D587e92.
1Current Steering Committee Chairs.
2Co-Chair.
