DRUG RESISTANCE MUTATIONS AND TRANSMISSION CLUSTERS OF THE HIV-1 CRF01_AE SUB-EPIDEMIC IN BULGARIA

Background. In Bulgaria the second most predominant HIV-1 strain after subtype B is CRF01_AE. Material and Methods. 270 HIV-1 polymerase (pol) CRF01_AE sequences collected between 1995–2019 were analyzed with the phylogenetic approach. To identify transmission clusters, we used two different genetic distance thresholds (d), i. e. 1.5% and 0.5%. Results. Genetic distance d=1.5% defined more distant infections in a huge cluster of 154 sequences composed mostly of people, who inject drugs (PWID), whereas when (d) was reduced to 0.5%, we determined more recent transmissions and the large cluster disintegrated into smaller ones. Different drug resistance mutations (DRM) were detected in patient’s HIV-1 pol sequences and were most common in male heterosexual (HET) single sequences. Conclusions. Our data showed repeated


INTRODUCTION
Acquired immunodeficiency syndrome (AIDS) is caused by two lentiviruses, human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) (1). HIV-1 is more prevalent in the world. The strains of HIV-1 can be classified into four groups: M (major), N (new), O (outlier) and P (2). Group "M" is the main group that is responsible for the global HIV-1 pandemic and contains at least 10 different subtypes (A-F, G, H, J, K and L), multiple circulating recombinant forms (CRFs) and various unique recombinant forms (URFs). HIV-1 subtype CRF01_AE was first isolated in Thailand and now predominates in South Asia and has spread throughout the world (3). The global prevalence of CRF01_AE is 5%, and it is responsible for up to 4% of the HIV-1 infections in Europe according to the SPREAD study (4). In previous studies, CRF01_AE was found to be one of the most disseminated clades in Bulgaria with around 20%, which is likely the highest percentage across Europe (5,6,7). Within the country, CRF01_AE is unevenly distributed and mainly affects the vulnerable population of PWID in the region of the capital of Bulgaria, Sofia, with 35% of all HIV-1 infections (8). With this study, we aim to determine the dynamics of distribution and transmission of CRF01_AE in Bulgaria. We intend to use epidemiological data and to link it ADDRESS FOR CORRESPONDENCE: Lyubomira Grigorova, 44A Gen. Stoletov Blvd., Sofia 1233, lyubomiragrigorova@gmail.com, tel.: +359 2 9318071 to the data obtained by sequencing the samples. Thus, we aim to clarify the events that have led to the transmission and production of resistance mutations. In addition, we will analyze HIVresistant mutations in this strain. We hope that our study results will help to develop better strategies for public health prevention in Bulgaria.

• Patient samples, research ethics and consent
Plasma samples were collected in the National Reference Confirmatory Laboratory of HIV (NRCL of HIV) of the National Center of Infectious and Parasitic Diseases (NCIPD) in the period 1995-2019. Demographic and epidemiological information was collected during the diagnostic process in accordance with national regulations. Through anonymous codes, blood samples, demographic and clinical information was linked following the ethical standards of Bulgaria (6). The Ethics Committee of NCIPD (NCIPD IRB 00006384) approved the study. • Sequence generation and analysis Viral RNA extraction was performed following the protocol of Abbott ViroSeqTM HIV-1 Genotyping Test v2.0 from serum or plasma. A fragment of the HIV-1 pol gene was generated using a ViroSeq HIV-1 genotyping test using an Applied Biosystems 3130xl genetic analyzer (8). HIV-1 subtypes were determined using Internet-based tools REGA HIV-1 subtyping tool version 3.0 (9) and COMET v2.3 (10). We used the Genotypic Resistance Interpretation Algorithm of the Stanford University HIV Drug Resistance Database (https://hivdb.stanford.edu/ hivdb/by-sequences/) to determine HIV-1 resistance mutations (DRMs) and their type. By using MUSCLE algorithm implemented in AliView version 1.23 (11,12) and MAFFT version 7 (13,14) sequences were aligned and some of the sequences were additionally manually aligned. The bioinformation program MicrobeTrace (15) was used to identify transmission clusters. Phylogenetic analysis was conducted with the Tamura-Nei algorithm (d) and clusters were determined at a genetic distance of 0.5% and 1.5%. Each pol sequence was represented graphically as a separate node. When two or more sequences were connected by certain threshold, they were labeled as a cluster, while nodes that did not connect to others were entitled as single sequences.

• Characteristics of the CRF01_AE subepidemic in Bulgaria
The first case of HIV-1 CRF01_AE infection in Bulgaria was detected in 1995 in a heterosexual man. The first mother-to-child transmission (MTC) was found 4 years later. The next important event in this sub-epidemic was the introduction of the CRF01_AE in the group of PWID in 2002. In 2009 in Sofia, 26 individuals were diagnosed with CRF01_AE, 18 (69.2%) of whom were PWID, indicating ongoing outbreak among PWIDs. In 2011, 31 cases were identified, 17 of whom PWID, 12 HET and 2 MSM. The total number of people diagnosed with HIV-1 subtype CRF01_AE by the end of 2019 was 270 or 16% of all patients with HIV. Analysis of gender distribution showed higher number of men than women. In CRF01_AE, men were 187 (69.3%) and women 83 (30.7%) with a ratio of 2.25:1, while the ratio found in other subtypes was with a higher weight of men 4:1. The age at diagnosis varied, youngest diagnosed was a newborn (0 years), the oldest was 63 years old. The largest share of those diagnosed was between 20-29 (40%), followed by 30-39 (33.3%) years of age, while the infected young (≤19) and older individuals (≥50) represented a smaller share. Patients indicating that the infection was acquired in Bulgaria were 251 (93%) and those infected abroad (mainly Western European countries) were 19 (17%). Analysis of the geographic distribution demonstrated that most of the infections were found in individuals from Sofia, 164 (60,7%), and the rest were dispersed across the country. When comparing transmission groups with CRF01_AE, we found that PWID were 141 (52.2%), followed by HET -101 (37.4) and MSM -13 (4.8) ( Table 1).

DISCUSSION
In 1995 CRF01_AE was identified, approximately 10 years after the first detected HIV-1 infection in Bulgaria. CRF01_AE was first introduced in HET and in 2002 the strain was transferred into the PWID community. CRF01_AE was rapidly disseminated among this population leading to a local outbreak in 2009 (6,8,7). Thereafter CRF01_AE became the second predominant subtype in Bulgaria and nowadays it is the most prevalent among PWID (52%) and least in MSM (4.8%). In addition, we found that a significantly higher number of women were infected with CRF01_AE (30.7%) than those infected with other HIV-1 subtypes (18,19 (16,20).
Fortunately, as in our previous HIV-1 studies in Bulgaria, most DRMs were found in cases that did not cluster or were in transmission pairs (7,17) and were much less prevalent in PWIDs (1.1%) compared to HET (28.7%) and MSM (23.7%).
The increased numbers of HIV-1 CRF01_AE infections in PWIDs in Bulgaria has led to a strong public response and measures by the Bulgarian Ministry of Health and NGOs (21). Campaigns have been put in motion to distribute free needles, syringes and condoms, free testing for HIV and hepatitis, and various educational initiatives. The study included only patients whose HIV-1 pol sequences were successfully obtained, thus excluding patients who had underwent a successful therapy and their viral load was low, as well as people who had not yet been diagnosed. These limitations may affect our analyzes and conclusions. Our study also included individuals who are likely infected abroad, this suggesting a more extensive study of transmission networks. To analyze the spread and transmission clusters of HIV-1 subtype CRF01_AE we conducted a phylogenetic analysis of the available HIV-1 pol gene sequences. Our study found transmission clusters indicating local outbreak among PWID. Resistance mutations were found mostly in HET and non-clustering singleton sequences. To better understand and control HIV-1 epidemic, continuous monitoring of this and other subtypes of HIV-1 in Bulgaria is further needed.