Rapid CD4+ T-cell decline is associated with coreceptor switch among MSM primarily infected with HIV-1 CRF01_AE in Northeast China : AIDS

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Rapid CD4+ T-cell decline is associated with coreceptor switch among MSM primarily infected with HIV-1 CRF01_AE in Northeast China

Cui, Hualua,b,c,*; Geng, Wenqinga,b,c,*; Sun, Honga,b,c; Han, Xiaoxua,b,c; An, Minghuia,b,c; Jiang, Yongjuna,b,c; Zhang, Zininga,b,c; Chen, Zhiweid; Xu, Junjiea,b,c; Hu, Qinghaia,b,c; Zhao, Bina,b,c; Zhou, Bennana,b,c; Shang, Honga,b,c

Author Information
doi: 10.1097/QAD.0000000000001981

Abstract

Introduction

The transmission of HIV-1 among MSM is rapidly increasing in China [1]. The CRF01_AE subtype is highly prevalent in Chinese MSM and accounts for more than 80% of HIV-1 infections in MSM in Liaoning Province of Northeast China [2,3]. A cross-sectional study reported lower baseline CD4+ T-cell counts in Shanghai MSM infected with CRF01_AE than with other subtypes [4]. A retrospective investigation showed a faster CD4+ T-cell decline in CRF01_AE-infected seroconverters, including 50% MSM, than non-CRF01_AE-infected patients in Singapore [5]. Likewise, chronically CRF01_AE-infected Chinese (MSM, 59.3%) were more likely to progress to AIDS than non-CRF01_AE-infected patients in another retrospective study [6]. In addition, a faster decrease in the CD4+ T-cell counts was observed in a Beijing MSM cohort (CRF01_AE, 53%) compared to MSM from resource-rich settings [7]. These reports indicate that CRF01_AE infection is likely to be responsible for not only high prevalence but also rapid CD4+ T-cell decline in Chinese MSM.

To date, however, little is known about the kinetic changes in the peripheral CD4+ T-cell counts among MSM since primary infection with HIV-1 CRF01_AE. Even less is known about the underlying virological mechanism. Viral tropism is known as a determinant for disease progression. Several recent studies predicted that CXCR4-utilizing/dual-tropic (X4/DM) viruses, rather than CCR5-utilizing (R5) viruses, were prevalent in newly diagnosed patients with HIV-1, suggesting that primary X4/DM viral infection may be responsible for the rapid disease progression in CRF01_AE-infected patients [4,6,8]. Because some studies mainly rely on sequence prediction, whether or not the situation is true in CRF01_AE-infected Chinese MSM remains to be carefully investigated. Dynamics of HIV-1 tropism during disease progression are distinct in disparate HIV-1 subtypes. For example, X4/DM viruses emerge in a fraction of HIV-1B-infected patients at late stages, whereas R5 viruses are dominant in most HIV-1C-infected patients throughout the infection [9]. Reports on coreceptor switch of CRF01_AE variants are scarce. Considering the essential role in disease progression, the dynamic change in coreceptor usage of CRF01_AE viruses needs to be clarified.

In the present prospective study, we investigated seroconversion in a large cohort of 1388 MSM at risk of HIV-1 infection in Liaoning Province of Northeast China. By following up a cohort of MSM primarily infected with HIV-1 CRF01_AE among the at-risk population, we aimed to determine their natural courses of disease progression and to identify possible underlying factors associated with the disease progression. Our results showed that patients with primary HIV-1 CRF01_AE infection underwent rapid CD4+ T-cell count decline without antiretroviral therapy (ART). R5 virus was found to be prevalent in MSM with primary CRF01_AE infection in Northeast China. However, high frequency of the patients experienced coreceptor switch within 3 years after infection and accelerated CD4+ T-cell loss. Our studies support the strategy of immediate treatment for CRF01_AE infection to prevent the rapid disease progression related to coreceptor switch.

Methods

Study design and participants

A long-term prospective follow-up study was conducted among an MSM cohort in Liaoning Province of Northeast China [10]. Inclusion criteria were a CRF01_AE subtype and primary HIV-1 infection (PHI) defined by viral acquisition within 180 days (Fig. 1). Follow-up ended at the earliest of ART initiation, death, or the last visit up until 31 May 2017. Informed consent was obtained before this study. The study protocol was approved by the Medical Research Ethics Committee of the First Affiliated Hospital of China Medical University.

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Fig. 1:
Study flow diagram for the present study.There were 1388 MSM with a high risk of HIV-1 infection in Liaoning Province, Northeast China, recruited in the present prospective study during December 2008 to July 2012. Screening of the eligible patients is shown. ART, antiretroviral therapy; CRT, coreceptor tropism; PHI, primary HIV-1 infection.

Identification of HIV-1 infection and estimation of the infection date

Blood samples were collected every ten weeks from MSM at risk of infection. HIV-1 infection identification was performed as reported previously [11]. The date of infection for an individual patient was estimated according to a self-reported date by the patient who was aware of an unambiguous exposure (Fig. 2a). To verify the estimated date of infection, Fiebig stages were defined by the detection of HIV-1 RNA, p24 antigen, and HIV antibody in plasma (Fig. 2b). The estimated date of infection well matched the Fiebig stages for each patient [12,13]. For patients without convincing self-reported date of exposure, the midpoint between the documented last negative and first positive test based on the HIV antibody test was adopted [14,15].

F2-2
Fig. 2:
An overview of the enrolled patients with PHI.(a) Estimated infection days. Each symbol represents a patient with PHI. The date of infection was estimated according to a self-reported date by the patient with a definite exposure time (●) or the midpoint between the last documented negative and first positive test based on the HIV antibody test (○). (b) Fiebig stages defined by HIV-1 clinical laboratory test results. IQR, interquartile range; ND: not done; PHI, primary HIV-1 infection; PID, patient identity.

Follow-up of patients with primary HIV-1 infection

Patients were enrolled in the study cohort starting at the diagnosis of PHI between December 2008 and July 2012, and followed until May 2017. During the follow-up period, there were weekly visits up to 2 months after infection (7 visits), monthly visits up to 6 months (5 visits), and quarterly visits after that (17–31 visits, if untreated). Peripheral blood was collected for CD4+ T-cell count and viral load measurements at each visit. Plasma antibodies against hepatitis B virus (HBV) and hepatitis C virus (HCV) were detected within 6 months after infection.

Genotypic prediction for coreceptor usage

A near full-length HIV-1 genome was acquired using a two-fragment method. Single genome amplification (SGA) of the 5-kb 5’ and 3’ half-genomes was performed as previous described [16,17]. The 3’ half-genome amplicons were sequenced at the C2-V5 region for coreceptor prediction, and a randomly selected amplicon was sequenced directly for full-length genome analysis using internal walking primers. Coreceptor usage based on the SGA sequences of the V3 region was predicted by Geno2pheno (http://coreceptor.bioinf.mpi-inf.mpg.de/index.php) with false-positive rates (FPRs) of 10, 5, and 2%, and the Position-Specific Scoring Matrix (PSSM) approach (http://indra.mullins.microbiol.washington.edu/webpssm/). X4/R5 (PSSMx4r5) and SI/NSI (PSSMsinsi) matrices for subtype B were used. In addition, a combination of criteria from the 11/25 and net charge rules was performed [18]. Genotypic prediction of coreceptor usage was determined using the first available sample within 180 days after infection.

Phenotypic determination of coreceptor tropism

Primary HIV-1 isolates were acquired by a co-culturing method using mixed peripheral blood mononuclear cells from HIV-1-infected patients and healthy donors. GHOST (3) cells expressing CD4+ and either CCR5 or CXCR4 were used as indicator cell lines [19]. U87 cell lines (U87.CCR5 and U87.CXCR4 cells) were also used for coreceptor usage determination in PHI [20]. Green fluorescent protein expression and supernatant p24 levels were analyzed after in-vitro infection on day 3 and day 7, respectively. Infectious molecular clones AD8 and NL4–3 were included as positive controls for CCR5 and CXCR4 usage, respectively. Phenotypic determination was performed by GHOST assay using sequential HIV-1 isolates. Date of coreceptor switch was estimated as the midpoint between the last R5 and first X4/DM virus detection based on phenotypic determination.

Immunological measurements

T-cell activation was determined by flow cytometry as previous described [21]. Human leukocyte antigen (HLA) class I genotyping was performed as previous described [22]. HLA characterization and calculated HLA score were analyzed as reported [23]. T-cell activation and HLA was characterized within 1 year after infection.

Statistical analyses

SPSS 17 (SPSS, Chicago, Illinois, USA) and Prism 5 (GraphPad Inc., La Jolla, California, USA) software were used to conduct statistical analyses. The rate of decline in CD4+ T-cell counts per month was calculated, with decreases being evaluated by means of linear mixed-effect models. Survival analyses were used for analyzing the expected time duration until coreceptor switch. P value less than 0.05 was considered statistically significant.

Results

Characteristics of study participants

To capture seroconversion of MSM, we frequently followed 1388 MSM at risk of HIV-1 infection in Liaoning Province of Northeast China during December 2008 to July 2012. Eighty-nine patients were enrolled within median 8 days [interquartile range (IQR) 4–19] after diagnosis of PHI. After excluding the patients who did not meet the inclusion criteria, there were 3, 20, 17, 11, and 8 patients recruited, respectively, from 2008 to 2012, and followed until May 2017. A total of 59 eligible MSM with primary HIV-1 CRF01_AE infection were recruited in the present study (Fig. 1). The basic information, including age at infection, ethnicity, Fiebig stages, estimated infection time, CD4+ T-cell counts, and viral load at enrollment, HBV and HCV antibodies are summarized in Table 1. The majority of participants were 33 years old (IQR 24–42) from the Han population without HBV or HCV coinfection. The median estimated infection time at enrollment was 31 days (IQR 25–59) after infection.

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Table 1:
Characteristics of HIV-1 CRF01_AE-infected Chinese MSM.

Rapid decline in CD4+ T-cell count among MSM with CRF01_AE infection

The CD4+ T-cell levels of the 59 enrolled MSM were followed for a median of 3.2 years (IQR 1.9–4.4), and 188.2 person-years were accrued as described in the “Methods” section. The median number of total observations since PHI was 13 per patient (IQR 8–18). Seronegative MSM at risk of HIV infection in the long-term prospective follow-up cohort were randomly selected to reflect the CD4+ T-cell levels before HIV seroconversion in patients with PHI (285 MSM, unpublished data). The median CD4+ T-cell count of the seronegative MSM was 712 (IQR 564–902) cells/μl, which was similar to the reported normal value (734; IQR 368–1632) in healthy Chinese adults [24]. Unexpectedly, 45 cases (76.3%) among the 59 MSM with primary HIV-1 CRF 01_AE infection were subjected to a CD4+ T-cell count decrease to less than 350 cells/μl during follow-up, with a median time of 0.6 years after infection (IQR 0.2–2.7; Supplementary Fig. S1a and b, https://links.lww.com/QAD/B339). A linear mixed-effect model showed the slope of CD4+ T-cell decrease among these MSM with PHI was −0.10√CD4+/month. The CD8+ T-cell count declination was defined as below a reported lower limit of the 95% reference interval in healthy Chinese adults [24]. The counts of WBCs and CD8+ T cells in the peripheral blood showed a slight alteration during following up. However, the majority showed progressively decreased CD4+ T-cell counts (Supplementary Fig. S1c–e, https://links.lww.com/QAD/B339). Thus, our data show that disease progression is very rapid among these CRF01_AE-infected MSM in Northeast China.

R5 HIV-1 variants are prevalent in MSM with primary CRF01_AE infection

To determine the viral tropism during PHI, we employed four widely used algorithms based on SGA sequences of the V3 region within 180 days after infection (Supplementary Fig. S2, https://links.lww.com/QAD/B339). To obtain enough SGA sequences in patients with low viral load or limited sample volume (21 patients), proviral DNA rather than plasma RNA was used, which was reported to be concordant in tropism prediction with plasma viruses in PHI by ultradeep pyrosequencing [25] and bulk PCR [18]. A total of 458 SGA sequences were derived from 59 individuals; 360 (78.8%), 412 (89.9%), 442 (96.5%), 442 (96.5%), 446 (97.4%), and 450 (98.2%) sequences were predicted to be R5 viruses using Geno2pheno (FPR = 10, 5, and 2%) and the other three algorithms, respectively. Among the 59 patients, R5 viruses rather than X4/DM viruses were found in 41, 51, and 55 cases (69.5, 86.4, and 93.5%) as predicted by Geno2pheno (FPR = 10, 5, and 2%, respectively). The frequency increased to 91.5–96.6% when the other three algorithms were used. Phenotypes were determined using 49 available primary isolates in PHI from the 59 patients. Forty-eight isolates (98.0%) were R5 variants, and only one X4/DM variant (2.0%) was found (Supplementary Table S1, https://links.lww.com/QAD/B339). Compared to phenotypic determination, genotypic prediction was likely to overestimate X4/DM proportion among these CRF01_AE variants. However, both genotypic prediction and phenotypic determination demonstrate that R5 viruses are predominant in these MSM with primary CRF01_AE infection in Northeast China.

Coreceptor switch among MSM within 3 years after infection

X4/DM tropism was reported to be a risk factor for disease progression. Despite extremely high frequency of R5 viruses in primary HIV-1 CRF01_AE infection, R5 to X4/DM switch was taken into account in the present study. Given obvious mismatching between genotype and phenotype, we used phenotype to determine the pace of coreceptor switch and further understand the role of coreceptor usage in CD4+ T-cell decline. Serial primary isolates were acquired from the patients to investigate the coreceptor tropism switch, because the results of phenotypic determination are exactly the same by GHOST cell lines and by U87 cell lines in PHI, we performed phenotypes of coreceptor usage by GHOST cell lines using subsequent isolates. Phenotypic determination was performed on a cumulative number of 54 MSM including five patients without phenotype results in PHI, who jointly contributed 130.8 person-years of study time and 18 switches to the analyses (Fig. 3, Supplementary Table S2, https://links.lww.com/QAD/B339). A total of 141 isolates were used for phenotypic determination. X4/DM viruses were found in four, 11, two, and one patient from the first to fourth year after infection, respectively. During follow-up, 14 and five cases were censored during the second and the third years after infection, respectively. Survival analysis indicated 7.9% of the patients harbored X4/DM strains within 1 year after infection, including a patient with X4/DM viruses in primary infection. The other 92.1% remained dominant with R5 viruses. However, the coreceptor switch occurred cumulatively in 32.8% of the patients within 2 years after infection, and 39.5% within 3 years after infection (Fig. 3a).

F3-2
Fig. 3:
Coreceptor switch and dynamic changes of the CD4+ T-cell counts in MSM with CRF01_AE infection.(a) Survival analysis of patients with R5 to X4/DM coreceptor switch. (b) Dynamic changes of CD4+ T-cell counts of patients that experienced coreceptor switch within 3 years after infection compared to the others.

Excess risk for rapid CD4+ T-cell decline among patients with coreceptor switch within 3 years after infection

At enrollment, the basic characteristics of the patients harboring X4/DM viruses in the subsequent 3 years did not differ from those remaining R5 phenotypes (Table 1). Since the high frequency of coreceptor switch, linear mixed-effect models were used to compare the rate of decline in CD4+ T-cell between patients with and without coreceptor switch. The patients with coreceptor switch within 3 years after infection underwent a faster CD4+ T-cell decrease compared to those without coreceptor switch (P < 0.001; Fig. 3b). No marked change was found after adjusting for age, baseline CD4+ T-cell count, and HLA-B*44 (Table 2). To clarify the relationship between coreceptor switch and the subsequent rate of CD4+ T-cell decline, a sensitivity analysis was performed. We excluded CD4+ T-cell counts before time of coreceptor switch for patients who experienced coreceptor switch, and also the CD4+ T-cell counts within 482 days after infection (the median time of coreceptor switch in our cohort) for the others. CD4+ T-cell counts of patients with coreceptor switch within 3 years after infection decreased at a median rate of 10 cells/μl per month (IQR 1.8–19.6), whereas CD4+ T-cell counts of those without coreceptor switch in 3 years after infection declined at a much lower rate (median 5.0, IQR 3.2–6.3 cells/μl per month). Linear mixed-effect models demonstrated CD4+ T-cell counts dropped significantly faster in patients with coreceptor switch compared to those without coreceptor switch (P = 0.003). Thus, we demonstrate the early coreceptor switch within 3 years after infection is significantly associated with rapid CD4+ T-cell decrease.

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Table 2:
Decline of CD4+ T-cell count in HIV-1 CRF01_AE-infected Chinese MSM, from linear mixed-effect models.

Virological and immunological factors among these HIV-1 CRF01_AE-infected MSM with rapid CD4+ T-cell decline

Considering that two genetic clusters of CRF01_AE strains have been previously found in our region [26], we determined whether rapid CD4+ T-cell decrease was favorably associated with a particular viral cluster. A linear mixed-effect model indicated the rate of CD4+ T-cell decline in the patients with ENV cluster 1 was not significantly different from those with cluster 2 (P > 0.05; Supplementary Fig. S3a and b, https://links.lww.com/QAD/B339). Similar to ENV genes, there was no significant association between HIV-1 clusters of near full-length sequences and the rate of CD4+ T-cell decline (P > 0.05; Supplementary Fig. S3c and d, https://links.lww.com/QAD/B339). Moreover, no difference in the CD4+ T-cell loss rate was observed among patients with a viral load of at least 100,000 copies/ml and those with a viral load below 100,000 copies/ml at enrollment by a linear mixed-effect model (P > 0.05; Supplementary Fig. S4, https://links.lww.com/QAD/B339).

T-cell activation during PHI has been reported to be associated with rapid disease progression [27]. We determined the levels of CD38 and HLA-DR expression in T cells at enrollment (36 out of total 59 patients). Linear mixed-effect models showed marginal association between CD4+ cell decline rate and level of CD4+CD38+HLA-DR+ and CD8+CD38+HLA-DR+ T cells mixed effect (P = 0.07 and P = 0.08, respectively; Supplementary Table S2, https://links.lww.com/QAD/B339).

Considering the potential association between certain HLA I alleles and disease progression [28], we determined the HLA I frequencies in our study participants. Using a linear mixed-effect model, we found that disease progression was not associated with generally reported protective and risk HLA alleles except HLA-B*44 (P = 0.03; Supplementary Table S3, https://links.lww.com/QAD/B339), an allele related to slow CD4+ T-cell decline [29]. However, this allele was not associated with disease progression after adjustment for other potential associated factors (P > 0.05; Table 2).

Discussion

Several cross-sectional and retrospective studies have previously demonstrated a low level of baseline CD4+ T-cell counts, fast CD4+ T-cell decline, and rapid AIDS development in CRF01_AE-infected MSM in China, Singapore, and Thailand [4–6]. To verify the actual disease progression in CRF01_AE-infected MSM since sero-positivity, we performed a prospective cohort study in Northeast China and identified 59 eligible patients in Fiebig stage II–VI at enrollment. In this cohort, a majority of HIV-1 CRF01_AE-infected MSM displayed less than 350 CD4+ T cells/μl during follow-up. This finding indicates the necessity of identifying the underlying mechanism of the rapid CD4+ T-cell decline among CRF01_AE-infected MSM.

CCR5 and CXCR4 are the two major coreceptors for HIV-1 entry into target cells. In general, R5 viruses have selective transmission advantages and make up the majority of transmitted and founder viruses [30]. Multiple factors may restrict X4 viral entry during HIV-1 transmission, which leads to a ‘gatekeeping’ mechanism [31–33]. However, a high frequency (32.4–55.6%) of the X4/DM tropism was reported in four recent studies on HIV-1 CRF01_AE-infected Chinese: one study on recently infected MSM [8], two studies on newly diagnosed patients [4,34], and one study on patients infected within 3–4 years [6]. These reports implied that patients with primary HIV-1 CRF01_AE infection might harbor X4/DM viruses. A study on recently infected Chinese MSM suggested that X4/DM viruses played a role in CRF01_AE transmission [35]. In the present study, however, R5 viruses were prevalent in PHI. Both genotypic prediction and phenotypic determination showed a low frequency of X4/DM viruses in these MSM with primary HIV-1 CRF01_AE infection. Subsequent phenotypic determination verified that 92.1% patients were dominant with R5 viruses at 1 year after infection, indicating that X4/DM viruses in most of the CRF01_AE-infected patients did not gain a growth advantage at least 1 year after infection. The frequency of R5 viral infection (41/59, 69.5%, Geno2pheno FPR = 10%) in our cohort is comparable with an observation in Singapore, which found that 76.9% (10/13) of newly CRF01_AE-infected patients were predicted to carry R5 viruses using bulk PCR (Geno2pheno, FPR = 10%) [36]. Phenotype determination also uncovered a very low frequency (0.8–1.4%) of X4/DM viruses in primary nonsubtype-B HIV-1 infection in France and Switzerland, respectively [37,38]. Furthermore, recent evidence from 102 patients with acute infection suggested that the R5 phenotype was a property of the transmitted virus per se and not one that evolved during early infection [12]. Taken together, our results provided evidence that the R5 viruses are responsible for the majority of transmission events in MSM with primary HIV-1 CRF01_AE infection.

The present study was the first longitudinal investigation on HIV-1 CRF01_AE viral tropism phenotype among patients with well documented seroconversion records. We exactly delineated the trajectory of X4/DM viral emergence following R5 viral transmission in HIV-1 CRF01_AE infection. In the present study, genotypic prediction showed overestimation of CXCR4 usage compared to phenotypic determination among these HIV-1 CRF01_AE-infected patients with primary infection, in agreement with other reports on CRF01_AE infection [39,40]. In consideration to the poor concordance with phenotypic determination, genotypic prediction was not employed in the subsequent determination of tropism for coreceptor switch. Notably, survival analysis showed 39.5% of the patients experienced coreceptor switch within 3 years after infection. Moreover, the coreceptor switch was significantly associated with a fast drop of CD4+ T-cell counts. Our results are in line with that X4/DM viruses are more pathogenic than R5 viruses, which are reported to be associated with fast disease progression [41,42]. In addition to the observed rapid CD4+ T-cell decline in the present study, X4/DM viruses were also related to potential poor CD4+ recovery, despite therapy [43,44]. Therefore, our results lend support to the strategy of immediate treatment in the CRF01_AE-infected MSM to interrupt the coreceptor switch, so as to avoiding rapid disease progress and possible deteriorated prognosis, despite ART. Immediate initiation of treatment would probably not give rise to emergence of X4/DM variants due to preferential X4/DM provirus elimination by ART [45–47].

Various viral and host factors are associated with disease progression in HIV-1-infected patients. In the present study, the rapid loss of CD4+ T cells was not significantly related to virus clusters or HLA genotypes. Older than 35 years was found to be a risk factor for CD4+ T-cell decline in our cohort, in accordance with previous studies, which suggested that age was associated with CD4+ T-cell decline [48]. T-cell activation was reported as a potential marker for the prediction of rapid progression [27]. We found that the level of CD4+CD38+HLA-DR+ and CD8+CD38+HLA-DR+ T cells had marginal association with a decrease in CD4+ T cells, and greater sample size was required to verify the association between the activated T cells and disease progression.

Although there was a large effort to collect samples, the sample size in the present study was still limited. We believe that a larger sample size should be required to confirm our observations, albeit a great challenge for any study based on a long-term prospective follow-up cohort of well documented, untreated patients with PHI. Convenience sampling was performed in this study, which might have potential selection bias. In addition, the numbers of SGA sequences were limited for some patients due to a low viral load or sample volume, despite the high homogeneity of viruses in PHI [25].

In summary, our results support the high prevalence of R5 viruses in primarily HIV-1 CRF01_AE-infected MSM in Northeast China. High frequency of R5 to X4/DM switch was found among these patients within 3 years after infection, which was associated with the fast decline of CD4+ T-cell counts. Our results support immediate treatment to withhold coreceptor switch among the CRF01_AE-infected MSM, which is in favor of avoiding X4/DM-associated rapid disease progression.

Acknowledgements

The study was supported by the mega-projects of national science research for the 12th Five-Year Plan [2012ZX10001-006], the National Natural Science Foundation (81273238) and Key Laboratory of Fundamental Foundation from Liaoning Education Department (LS201604).

We gratefully thank Dr Yuntao Wu of George Mason University for his careful review of this manuscript.

Authors’ contributions: The study was conceived and designed by H.S., W.G., and Z.C. Data acquisition and analysis were performed by H.C., H.S., X.H., M.A., Y.J., Z.Z., B.Z., and B.Z. J.X. and Q.H. were responsible for patient recruitment. H.C. and W.G. wrote the first draft. H.C., W.G., and H.S. contributed to the final version of the paper.

Conflicts of interest

All authors read and approved the final manuscript and contributed significantly to the paper with no conflict of interest.

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* Hualu Cui and Wenqing Geng are the co-first authors of this manuscript.

Keywords:

coreceptor switch; CRF01_AE; MSM; primary HIV-1 infection; rapid progression

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