HIV-1 epidemic in China is featured by the highly geographical and temporal diversity of epidemic patterns throughout the whole country. This study was the first cross-sectional molecular epidemiological research with large sample size in Heilongjiang of Northeast China. Large sample size allowed us to preliminarily understand the HIV-1 epidemiological features and trends in different high-risk populations, age groups and in the individuals infected by different genotype infections or in different immune status and thus to more comprehensively understand HIV-1 epidemic in Northeast China.
The first finding in the present study was that male-male sexual contact among MSM was the major route of HIV-1 transmission and CRF01_AE was the dominant HIV-1 genotype during 2010-2016. Unlike the situation in some European and American countries where HIV-1 epidemic initiated among MSM, few cases were reported among Chinese MSM until the year 2005. HIV-1 prevalence among MSM rapidly increased, from 1.4% in 2005 to 8% in 2015. Since 2010, MSM population has become the highest-risk group for HIV-1 transmission in China . The HIV-1 epidemic in Chinese MSM unevenly distributes across provinces and regions, highly concentrating in municipalities, provincial capitals or cities with large population sizes and fast economic growth, such as Beijing, Shanghai, Guangzhou .
Harbin is the capital of Heilongjiang province, one of three economic centers in Northeast China as well as one of cities that were affected by the first wave of HIV-1 expansion among MSM during 2006-2008 . HIV-1 prevalence among MSM population in Harbin rapidly increased from 1.0–9.5% between 2006 and 2011 [15, 16]. Our previous studies demonstrated that MSM became the highest-risk group for HIV-1 infection in Heilongjiang province during 2009-2012, accounting for 57.9% and 69.0% of new diagnoses in 2009-2010 and 2011-2012, respectively . In this study, we found MSM cases accounted for 66.3% of all new diagnoses during 2010-2016, consistent with our previous studies [7, 11], indicating the samples collected in this study were representative of HIV-1 infections in Heilongjiang. The proportion of MSM cases in Harbin was also similar with the cities in where HIV-1-infected Chinese MSM concentrate, such as Jilin (another northeast province), Beijing  and Shanghai . Importantly, although the annual new diagnoses continued to increase, the MSM proportion remained stable during this study interval, suggesting a stably growing period of HIV-1 infected MSM during 2010-2016 and the increasing risk of HIV-1 acquirement and transmission among MSM population in this city of Northeast China.
CRF01_AE genotype accounts for only 5% of global HIV-1 infections, and concentrates in Southeast Asia and China [19, 20]. In 1990s, CRF01_AE virus was introduced into China and rapidly spread throughout the whole country. Since 2007, CRF01_AE has become the most prevalent genotype in China except the northwest region. In Northeast China, more than half of HIV-1 infections were caused by CRF01_AE genotype . Several sporadic studies provided limited epidemiological feature of HIV-1 genotype in Harbin city. Our previous studies reported that subtype B was the main genotype in 1997-2000 and in 2007 [8, 9] and that CRF01_AE surpassed subtype B and became the dominant genotype in 2011-2012 . In the present study, we found since 2010, CRF01_AE had become the main genotype in this region, and continued to dominate HIV-1 genotype during 2010-2016. Similar with the situation of HIV-1 infected MSM, the proportion of CRF01_AE cases in annual new diagnoses kept relatively stable (53.6%-66.2%), suggesting CRF01_AE epidemic in Harbin has also reached to a stably growing period. Furthermore, in the present study, near 70% of CRF01_AE infected participants acquired HIV-1 via male-male sexual contact, higher than the national average level (approximately 58%) , supporting the observation that CRF01_AE viruses (clusters 4 and 5) epidemic in Northeast China mainly concentrated in MSM population [12, 23, 24]. Recent studies demonstrated CRF01_AE cluster 4 has had a longer growth stage (> 10 years) than the other clusters  but is just in the very early stage of its epidemic in Northeast China . It was also found that CRF01_AE cluster 4 (rather than cluster 5) infection among Chinese MSM was associated with fast decline of CD4 count and poor immune recovery under combination ART outside of Northeast China [27, 28]. However, the pathogenic feature of CRF01_AE clusters in Northeast China is unknown, which will be one interesting issue in our future studies.
The second finding of this study was that new HIV-1 diagnoses exhibited a younger trend in this seven-year study interval, which was mainly attributed to the rapid expansion of CRF01_AE-infected young MSM. In 2005-2008, the clustering hotspots of Chinese young people living with HIV/AIDS (15-24 years old) mainly distributed within heterosexuals and intravenous drug users in southwest provinces. After 2008, new hotspots among MSM in central and northeast provinces emerged. On the national scale, new HIV-1 cases aged 15-24 years increased by an annual average of 35% during 2011-2015 . For Harbin city, HIV/AIDS cases aged 15-24 years increased about 5-fold between 2005 and 2012 . This study found the proportion of new diagnoses aged < 30 years and aged 15-24 years rapidly rose from 16.4% and 5.0–29.3% and 11.5% during 2010-2016, respectively, implying that young people have become a new vulnerable group of HIV-1 infection in Northeast China. More concerns should be put on this population in future.
CRF01_AE infections accounted for 64.3% (429/667) of newly diagnosed MSM in Harbin, slightly higher than the national level (57.36%) reported in a meta-analytic integration of 66 molecular epidemiological studies conducted during 2008-2016 . A study on HIV-1 infected MSM from 13 Chinese provinces indicated the proportion of CRF01_AE cases aged 16-25 years decreased from 55.4–43.5% between 2009 and 2014 . However, in the present study, both of the proportions of CRF01_AE cases aged < 30 years and aged 15-24 years among HIV-1 infected MSM quickly increased approximately three times during 2010-2016 (13.6–34.0% and 4.5–13.6%, respectively), suggesting a rapid-expansion period of CRF01_AE-infected young MSM cases in Harbin city. The measures for HIV-1 prevention and control targeting young MSM population should be strengthened.
The third finding was that proportion of new HIV-1 diagnoses with fine immune status (CD4hi cases) significantly increased in Harbin, and age, sexual transmission mode of HIV-1 infection and virus genotype were associated with the immune status of HIV-1 infected participants. The overall improved immune status could be partially explained by the scale-up of HIV testing in China. It was reported that between 2009 and 2018, the total person-times of HIV testing increased from 55.6 million to more than 240 million in China , which allowed finding out more cases at the early stage of the disease. Young people seem to be more likely to know their HIV-1 status. Compared with old people, young MSM had a higher HIV-1 incidence and a higher prevalence of HIV-1 recent infection . These studies support our finding that both younger in age and HIV-1 infection among MSM were positive factors associated with being CD4hi cases. However, it is worth noting that recently infected HIV-1 cases are usually highly infectious because of their high viral load, but they mainly remain undetected . And, young MSM exhibited a higher prevalence of high-risk behaviors (recreational drug use, unprotected anal intercourse and concurrent multiple sex partnerships) than old MSM . Therefore, recently infected young MSM will speed up HIV-1 transmission and secondary infection within MSM population. We speculate that the rapid increase of cases with high CD4 count in young MSM would continue to be an important feature of HIV-1 epidemic in Northeast China in following years.
HIV-1 genotype was identified as another factor associated with immune status of participants in this study. Several cohort studies in China have demonstrated that HIV-1 genotype was the independent variable associated with disease progression. CRF01_AE infection is commonly associated to lower baseline CD4 count, more rapid CD4 count decline and faster progression to immunodeficiency than non-CRF01_AE infection in China [34–36]. Consistent with the above findings, in this study, relative to non-CRF01_AE infection, CRF01_AE infection in Harbin had significantly lower odds of being CD4hi case.
In this study, 07&08&C infected cases kept stable increase during 2010-2016 (Table 1). More importantly, the risk group and age compositions of 07&08&C and CRF01_AE infected cases were quite similar (data not shown), suggesting the co-circulation of CRF01_AE and 07&08&C strains in similar population in Harbin. Extensive studies have demonstrated CRF07_BC infection exhibits a slow disease progression . Consistent with this, our data indicated both median CD4 count and the proportion of CD4hi cases in 07&08&C cases were much higher than those in CRF01_AE cases. The weaker pathogenicity of 07&08&C virus might make potential contribution to the increase of CD4hi cases. This was supported by our data from logistic regression analysis that relative to CRF01_AE infection, 07&08&C infection had higher odds of being CD4hi case. More importantly, the co-circulation of CRF01_AE and 07&08&C strains in Northeast China would augment the potential recombination between these viruses and subsequently promote the emergence of new recombinant HIV-1 forms , leading to new and larger burden on the prevention and control of HIV-1 in China.
One more evidence for the overall improved immune status of the participants in Harbin city was also observed in this study. As shown in Table 1, the proportion of “late diagnoses” (CD4 count < 200 cells/µl at diagnoses) in newly identified HIV-1 cases ranged from 20.7–23.1% in Harbin during 2010-2016, much lower than the national level (35.5%-41.8%) in 2010-2014  and the provincial level of Heilongjiang in 1993-2012 (28.6%-38.4%) . This may be due to the high availability of HIV testing in China and people's higher pursuit of health and life quality.
There were several limitations in this study. First, although we had made great efforts to collect samples from clinical monitoring sites, the sample size in early time period (2010-2011) was still small (n = 140). Therefore, in some statistical analyses, differences among groups or subgroups may be underestimated. Even so, several important findings were yielded. We believe that using a larger sample size will further confirm our findings. Second, in this study, we hypothesized that the incubation period and the interval between infection and diagnosis were same in different age groups, high-risk populations and HIV-1 genotype infected groups. Although this is generally true for most HIV-1 infected people, there might be some exceptions. Third, HIV-1 genotyping based on gag and env gene regions might introduce some bias. Further analyses based on the near full-length genome of HIV-1 will be required in future, but considerable labor and financial resources will be required at same time. Despite these limitations, we still believe our work could provide several clues for further research and HIV/AIDS control in Northeast China.