Although subtype B is the dominant HIV-1 subtype in western countries, there has been increasing circulation of non-B subtype strains in Europe . In Italy, the proportion of subtypes that were non-B was reported to be as low as 1.9% before 1997, but since that time this proportion has tended to increase, ranging from 4.7 to 12.6% [2–5]. Most non-B subtypes and circulating recombinant forms (CRF) have been found in heterosexual contacts, whereas among injecting drug users (IDU) the proportion of non-B subtypes and CRF remains very low .
Herein we describe an outbreak of subtype G among IDU in northern Italy newly infected with HIV. The samples were collected for the Italian Seroconversion Study (ISS), a prospective cohort of HIV seroconverters; the inclusion criteria for the ISS are the availability of a documented seronegative HIV test result, followed by a seropositive result within 12 months .
We analysed sequences from a subgroup of ISS participants recruited from five clinical centres, located in Milan, Turin and Rome. The subgroup consisted of 139 individuals (6.7% of the entire ISS cohort). Of them, 25 (18.0%) were IDU, 63 (45.3%) homosexual men, 47 (33.8%) heterosexual contacts, and four (2.9%) belonged to other/unknown exposure categories.
Protease and reverse transcriptase pol sequences were generated after RNA extraction, reverse transcriptase–polymerase chain reaction amplification, and automatic sequencing. A commercially available assay (ViroSeq v2 kit; Abbott Laboratories, Abbott Park, Illinois, USA) was used. The nucleotide sequences used in this study have been submitted to Gene-Bank (National Center for Biotechnology Information) under the following accession numbers EF219434 to EF219447.
All of the HIV-1 pol sequences were analysed using the REGA HIV-1 subtyping tool .
All G subtype sequences of the HIV-1 pol gene present in the HIV database (http://hiv-web.lanl.gov/content/index) were aligned with our 11 sequences (691 base pairs long), partial protease and partial RT region), using a subtype B reference sequence (B.US.98.1058) as the outgroup; the alignment was then manually edited using the Bioedit program . The maximum likelihood phylogenetic tree was generated with the Hasegawua, Kishino and Yano (HKY-85) nucleotide substitution model under gamma distribution, using a neighbour-joining tree (Jukes–Cantor distance) as a starting tree . The evolutionary model was chosen as the best-fitting nucleotide substitution model, according to the hierarchical likelihood ratio test implemented in the Model Test V3.0 software . The statistical robustness and reliability of the branching order in the maximum likelihood phylogenetic tree was confirmed with a bootstrap analysis using 1000 replicates and with the zero branch length test. All calculations were performed with PAUP*4.0 software written by Swofford .
As expected, most participants were infected with subtype B (112 individuals, 80.6%). Non-B subtypes were more frequently detected among IDU (48.1%) than among homosexual men (25.9%) or heterosexual contacts (22.2%). The majority of non-B subtypes were subtype G (12 individuals, 9%), or undetermined (10 individuals, 7%).
Surprisingly, all G subtypes but one (11 of the 12 isolates) were found among IDU (the remaining individual was a woman belonging to the category of heterosexual contacts), and all of them were from Turin (north-western Italy); only one of them was not Italian (i.e. a man from Tunisia). The proportion of HIV-1 G subtype strains isolated after 1998 in Turin was 34.3%, of which 50% were isolated from individuals who seroconverted only in 2004, suggesting an increase over time of such a subtype in the northen Italian city. Overall, this subtype represented 64.7% of all strains identified among IDU between 1999 and 2004 (data not shown).
The phylogenetic analysis (Fig. 1) indicated that the outbreak was of monophyletic origin and was caused by HIV-1 strains that were similar to those from western Africa . The phylogenetic relationships were supported by the bootstrap analysis, which produced a value of 100% between the Italian G subtypes, 85% between the Italian G subytpes and one of thewestern African strains (accession no. AY181075); the relationships were also confirmed by zero branch length test (P < 0.001).
This is the first report of an outbreak of HIV-1 subtype G among IDU in Italy. This was largely unexpected, because most infections reported among Italian IDU are caused by subtype B. In a previous study, all sequences from 145 European IDU, including 20 Italian IDU from Turin, belonged to subtype B , whereas few HIV-1 non-B clades were detected among IDU in Finland, Spain, and Switzerland [13–16]. Large epidemics of subtype A have occurred among IDU in several states of the former Soviet Union, and a new epidemic caused by a CRF03 A/B was reported among IDU in northern Russia .
With specific regard to subtype G, this has been detected only rarely in Italy, and only among heterosexual contacts [2,5]. Similarly, in other countries in the same geographical area (e.g. France), none of the identified G subtypes were detected among IDU .
The monophyletic origin of this outbreak probably indicates a single point of introduction. Although the likely source was not identified, our strain was similar to those from west Africa (i.e. Nigeria). The hypothesis that the introduction of the subtype is related to drug trafficking conducted by west African immigrants remains to be tested.
In conclusion, this outbreak of HIV-1 subtype G among Italian IDU suggests that viral diversity is also increasing in population groups that had been exclusively characterized by subtype B. The monophyletic origin and the explosive dynamic of this outbreak can be attributed to the efficiency of blood transmission among IDU. This report suggests that the introduction of different subtypes in a specific risk population may give rise to future epidemic waves of unpredictable outcome, highlighting the importance of continuing molecular surveillance of the HIV-1 non-B strains in Italy, as well as in other western European countries.
The authors would like to thank Mark Kanieff for linguistic revision of the manuscript.
Sponsorship: This research was supported by a grant from Programma Nazionale Ricerche AIDS – Progetto Epidemiologia, Istituto Superiore di Sanità.
The first and the second authors contributed equally to the work.
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