From November 2011 to May 2012, a total of 2851 patients were recruited from a hospital in Vientiane, the capital of Laos, with a mean age of 32 years old. Most of these patients were Lao Loum ethnicity (85.8%), 58.8% of them were female, and 59.0% of them domiciled in Vientiane (Table 1).
Variables Total patients HIV positive Chi square P value Gender 0.018 0.894 Female 1675 40 (2.4%) Male 1176 29 (2.5%) Ethnicity 4.201 0.122 Lao Loum 2445 65 (2.7%) Lao Soung 264 3 (1.1%) Lao Theung 142 1 (0.7%) Domiciles of origin 14.514 0.024 Vientiane Capital 1682 54 (3.2%) Khammouan 72 2 (2.8%) Bolikamxai 517 10 (1.9%) Xaignabouli 57 1 (1.8%) Xiangkhoang 129 1 (0.8%) Vientiane 209 1 (0.5%) Other provinces* 185 0 *Attapu, Bokèo, Champasak, Houaphan, Louang Namtha, Louangphrabang, Oudômxai, Phoôngsali, Salavan, Savannakhet, Xékong
Table 1. The prevalence of HIV among hospital patients in Vientiane Prefecture of Laos.
According to the results of the amplification of the HIV gag fragment, 2.4% of the participants (69/2851) were found to be HIV proviral DNA positive (Table 1). The prevalence of HIV among patients who domiciled from different provinces was statistically distinct (P = 0.024). Patients domiciled from Vientiane had the highest HIV prevalence (3.2%) followed by Khammouan (2.8%) and Bolikamxai (1.9%). The HIV prevalence was not significantly different between female and male, ethnic Lao Loum, Lao Soung and Lao Theung (P > 0.05).
From the proviral of the 69 HIV-positive patients, 61 near full-length genomes of HIV were successfully amplified and sequenced. The results of the Recombinant Identification Program that is available in HIVDB showed that 56 sequences were CRF01_AE, and the other five sequences were recombinants form of HIV subtype B and CRF01_AE. Furthermore, the ML tree showed that these 56 sequences clustered with CRF01_AE reference sequences, confirming that they were CRF01_AE (Fig. 2). The remaining five sequences did not cluster with any reference sequences and formed a unique cluster with 100% maximum parsimony bootstrap value, implying that they had a close genetic distance with each other and might be new HIV recombinant forms (Fig. 2).
Figure 2. The maximum likelihood tree of HIV near full-length genomes. The red spots indicate the sequences that amplified from patients in Vientiane Capital, Laos. The red and blue shadows indicate HIV sequences of CRF01_AE and the newly identified CRF97_01B, respectively. The support of each branch is shown at each node, and only bootstrap values greater than 75% are shown.
To explore whether these five sequences were new recombinant forms and shared the same breakpoints, bootscan analysis was performed using subtypes B, F, G, H, J, K and CRF01_AE as reference subtypes. Bootscanning plots confirmed that they were recombinant forms of two subtype B fragments and two CRF01_AE fragments and shared the same breakpoints (Fig. 3A). The independent ML tree of each segment further confirmed the results of the bootscan analysis (Fig. 3B). According to the HIV nomenclature proposal, these five sequences were designated to be CRF97_01B, being the first HIV circulating recombinant form that identified in Laos. CRF97_01B was constructed of HIV CRF01_AE from 227 to 1170 nt, 2790 to 8995 nt, and subtype B from 1142 to 2794 nt, 8951 to 959 nt (Fig. 3C).
Figure 3. The identification of HIV subtype CRF97_01B. A The bootscan plots of CRF97_01B. The lines with different colors indicate different HIV subtypes. B The maximum likelihood trees based on the sub-regions of CRF97_01B. Subtype references are downloaded from the Los Alamos HIV Sequence Database, including subtype B to K, CRF01_AE and a sequence of chimpanzee. The red circles indicate the sequences that amplified from patients in Vientiane Capital, Laos. The red and blue shadows indicate sequences of CRF01_AE and subtype B. The support of each branch is indicated at each node, and only bootstrap values greater than 75% are shown. C Schematic maps of CRF97_01B. The rectangles with different colors indicate different HIV subtypes.
To characterize the dynamics of CRF01_AE in Laos and its bordering countries, Bayesian phylogeographic analysis was performed based on long CRF01_AE fragments. The results of the ML tree showed that CRF01_AE in Southeast Asia first circulated in Thailand (Fig. 4). All sequences formed ten main clusters, and sequences from Laos were distributed in nine of them, implying that HIV in Laos originated from multiple lineages. Sequences of Laos mainly originated from Thailand, and only three sequences originated from China (Cluster 1 in Fig. 4). Additionally, the CRF01_AE segment of CRF97_01B was derived from sequences of Laos in the late-1980s (Cluster 2 Fig. 4).
Figure 4. The maximum clade credibility tree based on sequences of HIV CRF01_AE. HIV CRF01_AE sequences (2790–8995 nt according to HXB2) in Southeast Asia are used for Bayesian phylogeographic analysis. Sequences with different origins are shown with different colors. Clusters including sequences from Laos are shown with red typeface. The black spots indicate the sequences of the newly identified CRF97_01B. The black square indicates the node of CRF97_01B, and the number indicates the origin time of CRF97_01B.
Among these 61 near full-length genomes, seven contained drug resistance mutations (Table 2). The mutations included A62V, D30N, E138G, E138K, F116Y, G140R, G163R, G190E, G190S, G48R, G73S, M184I, M184V, M230I, M46I, Q151M, T215A, V75I and Y181I. Among these seven sequences, one had high-level resistance to nucleoside reverse transcriptase inhibitors (NRTI), nonnucleoside reverse transcriptase inhibitors (NNRTI) and protease inhibitors (PI); two had high-level resistance to NRTI and NNRTI; one had low-level resistance to NNRTI and middle-level resistance to PI; one had middle-level resistance to NNRTI; and two had low-level resistance to NRTI or integrase inhibitors.
Samples NRTI NNRTI PI IN Mutations Resistant Mutations Resistant Mutations Resistant Mutations Resistant VT36M3933 M184I ABC (L), FTC (H), 3TC (H) E138K, G190E EFV (H), ETR (M), NVP (H), RPV (H) D30N, M46I, G48R, G73S ATV/r (L), FPV/r (L), IDV/r (L), LPV/r (L), NFV (H), SQV/ r (L) VT38M3815 A62V, V75I, F116Y, Q151M, M184V ABC (H), AZT (H), D4T (H), DDI (H), FTC (H), 3TC (H), TDF (M) Y181I EFV (M), ETR (H), NVP (H), RPV (H) VT42M2998 M184I ABC (L), FTC (H), 3TC (H) G190S, M230I EFV (H), ETR (L), NVP (H), RPV (M) VT37F3781 T215A AZT (L), D4T (L) VT27F3026 E138G RPV (L) M46I NFV (M), BL50F5250 E138K RPV (M) VT30F3147 G140R, G163R EVG (L), RAL (L) NRTI Nucleoside reverse transcriptase inhibitors, NNRTI non-nucleoside reverse transcriptase inhibitors, IN integrase inhibitors, PI protease inhibitors, L low level resistance, M intermediate resistance, H high-level resistance, ABC abacavir, FTC emtricitabine, 3TC lamivudine, AZT zidovudine, D4T stavudine, DDI didanosine, TDF tenofovir, EFV efavirenz, ETR etravirine, NVP nevirapine, RPV rilpivirine, ATV/r atazanavir/r, FPV/r fosamprenavir/r, IDV/r indinavir/r, LPV/r lopinavir/r, NFV nelfinavir, SQV/r saquinavir/r, EVG elvitegravir, RAL raltegravir
Table 2. The drug resistance mutations of HIV sequences amplified from patients in Vientiane Capital, Laos.