HIV-1 transmission networks across Cyprus (2010-2012)

A molecular epidemiology study of HIV-1 infection was conducted in one hundred diagnosed and untreated HIV-1-infected patients in Cyprus between 2010 and 2012, representing 65.4% of all the reported HIV-1 infections in Cyprus in this three-year period, using a previously defined enrolment strategy. Eighty-two patients were newly diagnosed (genotypic drug resistance testing within six months from diagnosis), and eighteen patients were HIV-1 diagnosed for a longer period or the diagnosis date was unknown. Phylogenetic trees of the pol sequences obtained in this study with reference sequences indicated that subtypes B and A1 were the most common subtypes present and accounted for 41.0 and 19.0% respectively, followed by subtype C (7.0%), F1 (8.0%), CRF02_AG (4.0%), A2 (2.0%), other circulating recombinant forms (CRFs) (7.0%) and unknown recombinant forms (URFs) (12%). Most of the newly-diagnosed study subjects were Cypriots (63%), males (78%) with median age 39 (Interquartile Range, IQR 33–48) reporting having sex with other men (MSM) (51%). A high rate of clustered transmission of subtype B drug-sensitive strains to reverse transcriptase and protease inhibitors was observed among MSM, twenty-eight out of forty-one MSM study subjects (68.0%) infected were implicated in five transmission clusters, two of which are sub-subtype A1 and three of which are subtype B strains. The two largest MSM subtype B clusters included nine and eight Cypriot men, respectively, living in all major cities in Cyprus. There were only three newly diagnosed patients with transmitted drug resistant HIV-1 strains, one study subject from the United Kingdom infected with subtype B strain and one from Romania with sub-subtype A2 strain, both with PI drug resistance mutation M46L and one from Greece with sub-subtype A1 with non-nucleoside reverse transcriptase inhibitors (NNRTI) drug resistance mutation K103N.


Introduction
In the last twenty years, combined antiretroviral drug therapy (cART), has been developed to specifically target HIV-1 with outstanding success, resulting in a dramatic decrease in mortality among HIV-1-infected individuals. However, the genetic variability of HIV-1 constitutes a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 the most striking challenge in effectively treating HIV-1 infection. Specifically, the accumulation of drug resistant mutations during suboptimal therapy severely affects the clinical benefits of cART, leading to impaired therapy outcome [1][2][3] and the transmission of drug-resistant HIV-1 strains to newly-infected individuals in European countries [4][5][6][7][8], recently reported at just below 9% among newly-diagnosed individuals from 26 European countries between 2008 and 2009 [5]. Furthermore, according to the most recent molecular epidemiology study of HIV-1 infection in Europe, the most prevalent Group-M subtypes and inter-subtype circulating recombinant forms (CRFs) were subtype B (66.1%), followed by sub-subtype A1 (6.9%), subtype C (6.8%) and CRF02_AG (4.7%) with significant variances in subtype distribution among European countries, immigrant populations and patient risk-groups [9].
The first molecular epidemiological study for the HIV-1 infection in Cyprus, constituting the eastern European Union frontier in the Mediterranean Sea, was reported in 1995 [10]. HIV-1 was initially reported in Cyprus in the mid-1980s and the first reported HIV-1-infected patient in Cyprus was a young woman who reported living in the United States who was diagnosed in 1986 and died in 1987 [10]. Subsequently, the HIV-1 infection in Cyprus has been studied by densely sampled prospective molecular epidemiological studies of newly diagnosed patients (88% registered HIV-1-infected individuals until 2009) [11][12][13]. The main findings from the aforementioned HIV-1 molecular epidemiological studies in Cyprus is first, the high genetic heterogeneity of HIV-1 infection in the island as a result of a continuous influx of new HIV-1 strains from many countries, mainly from African countries, and second, the low transmitted resistance to HIV-1 antiretroviral drugs.
As part of our ongoing effort to monitor the genetic diversity of HIV-1 infection and the transmission of antiretroviral drug resistant HIV-1 strains in Cyprus, in this molecular epidemiological study we generated and analyzed HIV-1 pol sequences from one hundred HIV-1 diagnosed and untreated patients in Cyprus between 2010 and 2012 (65.4% of reported HIV-1 infections in Cyprus in this three-year period), using a previously defined enrolment strategy and previously established experimental procedures [11][12][13]. Furthermore, we examined the reported risk factors and other epidemiological information in an effort to gain further understanding into risks underlying the observed HIV-1 transmission networks in Cyprus during the three-year period, between 2010 and 2012.

Study subjects
For the period 2010 to 2012 blood samples were obtained from one hundred consenting HIV-1-infected individuals from the AIDS Clinic of Larnaca National Hospital, representing 65.4% of all the reported HIV-1 infections in Cyprus (area controlled by the Republic of Cyprus) in this three-year period. The blood samples from these individuals had been taken for standard genotypic drug resistance diagnostic purposes between January 2010 and September 2012 and were retrospectively added to this study after written consent from the study subjects as previously described [11][12][13]. Specifically, an informed consent form was signed by each subject and a questionnaire containing clinical, demographic and epidemiological information was filled by qualified medical personnel at the AIDS Clinic of Larnaca National Hospital. All samples and questionnaires were coded with a laboratory identifier number so as not to reveal the personal identity or the hospital registration identities of the study subjects. All blood samples were processed at the Laboratory of Biotechnology and Molecular Virology of the University of Cyprus within the same day of sampling. The majority of study subjects were Greek-Cypriots, although a number reported traveling or living abroad in the past. The HIV-1 sero-diagnosis of each subject was previously established by commercial enzyme-linked immunoassay and confirmed by Western blotting. All blood samples were processed at the Laboratory of Biotechnology and Molecular Virology of the University of Cyprus on the same day of sampling. This study is in conformity with regulations by the National Bioethics Committee and by the National Data Protection Commission in Cyprus.

PCR amplification of pol region (protease and partial reverse transcriptase) and determination of drug-resistant mutations
HIV-1 sequences encoding approximately 1461 nucleotides of the pol (protease, PR and partial reverse transcriptase, RT) region were amplified from each sample by reverse transcription nested PCR (RT-PCR) using plasma HIV-1 RNA. The detailed experimental method for the amplification of the pol region by RT-PCR using plasma HIV-1 RNA was described by Kousiappa et. al, [13]. As part of our previously established methodology, in each sample, the DNA sequences encoding the pol regions were determined by population sequencing using the second-round amplified PCR product as the template and two sequencing primers (2136 and 3462) [13]. DNA sequence reactions were performed by the BigDye Terminator Cycle Sequencing kit and sequenced with the ABI 3130 genetic analyzer (Applied Biosystems, Foster City, CA) according to the manufacturer's recommendations. HIV-1 drug resistance interpretation was defined based on the World Health Organization's (WHO's) list of drug resistance mutations for surveillance purposes, which was updated on 2009 [14]. Assessment of the levels of expected resistance to each of the three classes (nucleoside reverse transcriptase inhibitors, NRTI; non-nucleoside reverse transcriptase inhibitors, NNRTI; and protease inhibitors, PI) on the therapeutic response was predicted using the Stanford genotypic resistance interpretation algorithm (HIVdb version 8.3) [15] using the International AIDS Society-USA (IAS-USA) drug resistance mutation list, which was last updated on 2015 [16].

Phylogenetic analysis
HIV-1 subtyping was performed using the REGA HIV-1 & 2 automated subtyping tool (Version 2.0) [17,18] and confirmed by phylogenetic analysis using a global set of sequences (N = 216) representative of all pure HIV-1 subtypes and the most prevalent CRFs as references. Reference sequences were obtained from the Los Alamos database at Los Alamos National Laboratory (online), http://www.hiv.lanl.gov (last updated on 2017). Phylogenetic trees were reconstructed using approximately maximum likelihood (ML) method as implemented in the FastTree (version2.1) program [19] using the generalized time-reversible (GTR) model of nucleotide evolution with the "CAT" approximation for different evolutionary rates across sites [19]. To explore putative recombinant patterns in the viral sequences, we performed a bootscanning analysis using SimPlot (version3.5.1) [20]. Putative recombinants were confirmed by phylogenetic analysis using partial fragments showing discordant phylogenetic signals against a reference set of HIV-1 group pure subtypes obtained from the Los Alamos database. Bootscanning was performed with a sliding window of 400 nucleotides overlapped by 50 nucleotides to define the recombinant assembly. Details about identification of the phylogenetic clusters have been described previously [21]. In brief, phylogenetic clusters were defined as those with bootstrap support greater than 85% (phylogenetic confidence criterion), and with a mean genetic distance of fewer than 0.015 nucleotide substitutions per site.

Results
Eighty-two study subjects were newly diagnosed and blood was drawn within one month of HIV-1 diagnosis. The remaining eighteen patients were either chronically-infected (seven patients were diagnosed before 2009 and three in the three-year period 2010 to 2012) or with unknown date of HIV-1 diagnosis (eight patients). Study subjects were retrospectively enrolled in this study after written consent followed by the submission of a questionnaire containing clinical, demographic and epidemiological information that was filled by qualified medical personnel at the AIDS Clinic of Larnaca National Hospital. Patients who did not wish to participate were not asked to provide a reason for choosing to do so. The staff who completed the questionnaire did not report specific patient group(s) (i.e. gender, sexual orientation, IV drug use etc.) who were unwilling to participate in the study. A detailed description of the clinical profile of each patient is presented in Table 1. Epidemiological, demographical and clinical data were collected from each consenting study subject as previously described [5,7,[11][12][13].
The main characteristics of the study subjects are presented in Table 2. Seventy-eight individuals out of the one hundred participating individuals (78%) are male and the remaining twenty-two (22%) are female with the median age of 39 years (IQR, 33-47). Sixty-three subjects were Cypriot citizens living permanently in Cyprus at the time of study, even though a number of them reported traveling or living abroad in the past, whereas the remaining thirtyseven subjects were born abroad: Romania (twelve study subjects); Greece (five); Cameroon (five); United Kingdom (three); Ukraine (three); Bulgaria (two); Kurdistan (two); Mali (two); Togo (one); Lithuania (one) and Serbia (one). The most common reported risk factor of HIV-1 transmission was homo/bisexual contact (HBC) (51%) followed by heterosexual contact (HC) (33%), other risk factors (4%), unknown risk factor (6%) and not available data (6%). Investigation for other sexually transmitted diseases showed that three study subjects were infected by the herpes simplex virus (HSV), one patient with syphilis and two patients were diagnosed with anal warts (condyloma acuminatum) which are caused by the human papilloma virus (HHV). Furthermore, one study subject (1%) was infected with hepatitis B virus (HBV) and two others (2%) with hepatitis C virus (HCV). At the time of HIV-1 sero-diagnosis, the median CD4 count and the plasma virus load were 424 cells/μl (IQR, 223-653) and 4.41 log copies/ml (IQR, 4.09-4.91), respectively. Analyses of the HIV-1 pol sequences (nucleotide positions 2253 to 3359 of the HXB2 genome) indicated that subtypes B and A1 were the most common subtypes present and accounted for 41% and 19% respectively, followed by subtype F1 (8%), C (7%), CRF02_AG (4%), A2 (2%), other CRFs (7%) and unknown recombinant forms (URFs) (12%). The sequences obtained in this study were submitted to the Genbank and the accession numbers (pol sequences, KJ635931 -KJ636030) will be available prior to the publication of the manuscript.
A summary of the characteristics of patients with transmitted drug resistance mutations is shown in Table 3. The overall prevalence of transmitted drug resistance mutations (TDRM) to current HIV-1 antiretroviral drugs among the newly-diagnosed and drug-naive patients of the study cohort was 3.7% (3 out 82 study subjects). All of them were infected with viruses carrying a single TDRM. Specifically, there was one patient (CY369) infected with an HIV-1 strain with a non-nucleoside reverse transcriptase inhibitor (NNRTI) resistance mutation (K103N) and two patients (CY274 and CY314) with a protease inhibitor (PI) resistance mutation (M46L). CY369 is a 29-year old man born in Greece, infected with a sub-subtype A1 HIV-1 strain within the reported HBC transmission risk group. CY274 is a 53-year-old male born in the United Kingdom, infected with a subtype B strain within the reported HC transmission risk group and CY314 is 47-year-old female born in Romania, infected with a sub-subtype A2 strain and unknown transmission group. Additionally, there were two chronically sub-subtype F1 HIV-1-infected individuals from Romania (>52 weeks since diagnosis) with multiple drug resistant mutations. Study subject CY289, a Romanian citizen living in Cyprus, was infected with a sub-subtype F1 dual-class RT resistant strain carrying both K219Q and K103N mutations. The second study subject, also a Romanian citizen living in Cyprus, was also infected with a sub-subtype F1strain dual-class RT and PI resistant strain carrying D67N, K70R, (Continued) (Continued)  Fig 1B, Cluster 11).

HIV-1 infection in Cyprus
M184V, T215F, Y181C, M46L, I54V and V82F mutations associated with drug resistance. Both study subjects reported, however, that they were diagnosed HIV-1 seropositive in Romania prior to their arrival in Cyprus and both of them were receiving cART in Romania (Table 1). Therefore, these patients were not classified as drug naïve, and consequently, the identified resistance mutations associated with these patients were excluded from the transmission of HIV drug resistance analysis. Phylogenetic analyses, presented in the Fig 1A, discovered twelve phylogenetic transmission clusters highly supported by the set bootstrap support and the genetic distance criteria. Four of the clusters (Fig 1B: Cluster No3, No4, No5 and No6) included individuals infected with sub-subtype A1 strains, five clusters (Cluster No7, No9, No10, No11 and No12) with subtype B, one cluster with sub-subtype F1 (Cluster No1), one cluster with CRF02/B (Cluster No2) and one cluster with A/B recombinant strains (Cluster No8). The clinical and epidemiological information for all the individuals identified in the phylogenetic clusters are presented in Table 1.
Sub-subtype A1 clusters included: i) a heterosexual couple from Ukraine who were living in Cyprus since 1994 (No3), ii) a heterosexual couple from Greece (male) and Romania (female) who reported living in Cyprus since 1990 and 2007, respectively (No4), iii) four Cypriot men,  One of them reported HIV-1 contraction was likely contracted in Thailand or Russia by unprotected sex with a transsexual person. The other three men involved in this cluster reported that HIV-1 was likely contracted in Cyprus, two of them by unprotected sex with anonymous person and one of them with his partner who is not a study subject. Subtype B cluster No9 involved two men from Cyprus. One of them was a divorced thirty-year-old man who reported that HIV-1 infection was likely contracted in Greece by unprotected heterosexual contact, whereas the other one was a thirty-five-year-old married man who reported that HIV-1 infection was likely contracted in Cyprus by unprotected heterosexual contact with an anonymous person. The largest subtype B cluster, No10, involved nine men (six from Cyprus, one from Greece and one from Serbia) all of whom were MSM. Five of them reported that HIV-1 was contracted in Cyprus and one in Syria. Among the study subjects found in this cluster, there were two reported homosexual couples. The first couple consists of the study subjects CY276 and CY277, and the second one of CY346 and CY347. Furthermore, it was reported that the study subject CY349 is the homosexual partner of CY267, who was included in a previous molecular epidemiology study [11]. The median age of the study subjects found within this cluster is 32 years. Additionally, it was reported that four of them were living in the city of Limassol and three in Nicosia, whereas only two subjects were living in the rural areas of Nicosia and Limassol (one in each area). Subtype B cluster No12 is the second largest cluster identified in this study and it is consisted of eight men (seven from Cyprus and one from Greece). Six of them reported that the most likely route of infection was a hetero-bisexual contact, whereas two of them a heterosexual contact. Six of them reported that HIV-1 was contracted in Cyprus. The median age of the study subjects found within this cluster is 38 years. It was also reported that seven of them were living in rural areas of Lefkosia (two), Larnaca (one), Lemesos (one), Pafos (two) and Ammochostos (one), whereas only one subject was living in the city of Lefkosia. The geographical distribution of study subjects involved in clusters No10 and No12 are shown in Fig 2. Subtype B cluster No11 consisted of a heterosexual married couple (study subjects CY338 and CY339) from Cyprus. The wife (CY339) reported that she contracted HIV-1 infection in Cyprus by unprotected heterosexual contact with her husband (CY338).
Sub-subtype F1 cluster No1 involved a heterosexual married couple from Kurdistan, CRF02/B cluster No2 consisted of two Cypriot men (one of them reported himself as MSM),

Discussion
In this prospective study we are presenting the molecular epidemiology of HIV-1 infection and TDR in newly diagnosed patients in Cyprus between 2010 and 2012. Eighty-two newly diagnosed untreated patients participated in this study by providing demographic, epidemiological and behaviour information. This number represents the 53.6% of the antiretroviral naïve newly diagnosed patients, reported at the AIDS Clinic of the Larnaca General Hospital in Cyprus the period 2009-2010. The subjects were predominantly young Cypriot men reporting having sex with other men (MSM). The non-Cypriot subjects were mostly young people from Eastern European and African countries. Previous molecular epidemiology studies conducted in Cyprus from 1986 to 2009 have shown that the predominant subtypes were subtypes A, B, C and CRF02_AG [11][12][13]. Our HIV-1 infection in Cyprus study reveals that subtypes B and A1 were the most common subtypes in Cyprus and accounted for 41.0 and 19.0% respectively, followed by subtype C (7.0%), F1 (8.0%), CRF02_AG (4.0%), A2 (2.0%), other CRFs (7.0%) and URFs (12%). Significantly, twenty-eight out of forty-one MSM study subjects (68.0%) were implicated in five transmission clusters, two of which were consisted of sub-subtype A1 and three of subtype B strains. The two largest MSM subtype B clusters included nine and eight Cypriot men, respectively, living in all major cities and rural areas in Cyprus (area controlled by the Republic of Cyprus). Furthermore, our findings indicate that within the study cohort of newly diagnosed drug naïve subjects, the prevalence of TDR to current HIV-1 antiretroviral drugs was only 3.66%, which is significantly lower than known prevalence rates of TDR in newly diagnosed patients in other European countries [5].
In conclusion, the results of the present study indicate that the HIV-1 infection in Cyprus is increasingly identified among young Cypriot MSM being infected in Cyprus. By comparing the results of previous HIV-1 transmission studies in Cyprus conducted between 1986 and 2009, which showed that new HIV-1 infections were more frequently detected among immigrants mostly from African countries, the current results point to a changing HIV-1 transmission dynamic in the island. Also, the TDR remains consistently low (4%) in Cyprus in comparison to Europe where it is stable at around 8% [5].

Sequence data
GeneBank accession numbers for the sequences obtained in this study are as follows: pol sequences, KJ635931 -KJ636030.