BackgroundHIV-1 non-B subtypes have recently entered Western Europe following immigration from other regions. The distribution of non-B clades and their association with demographic factors, over the entire course of the HIV-1 epidemic, have not been fully investigated in Italy. MethodsWe carried out a phylogenetic analysis of HIV-1 pol sequences derived from 3670 patients followed at 50 Italian clinical centres over nearly three decades. ResultsOverall, 417 patients (11.4%) carried non-B subtypes. The prevalence of non-B strains increased from 2.6% in 1980 -1992 to 18.9% in 1993) in a subset of 2479 subjects with a known year of diagnosis. A multivariate analysis on a subset of 1364 patients for whom relevant demographic data were available indicated that African ethnicity, heterosexual route of infection and year of diagnosis were independently associated with non-B HIV-1 infection (P 0.0001). All pure subtypes, except for clade K, and seven circulating recombinant forms were detected, accounting for 56.6 and 34.1% of the non-B infections, respectively. The F1 subtype was the most prevalent non-B clade among Europeans and was acquired heterosexually in half of this patient population. Unique recombinant forms accounted for 9.4% of the non-B sequences and showed a B/F1 recombination pattern in one-third of cases. ConclusionsThe circulation of non-B clades has significantly increased in Italy in association with demographic changes. Spread of the F1 subtype and B/F recombinants appears to predominate, which may result in a redistribution of the relative proportions of the different strains, and this could lead to overlapping epidemics. Thus, the HIV-1 landscape in Italy may in future be distinct from that of the rest of Europe. IntroductionNine discrete lineages of group M HIV-1 (A-D, F-H, J and K) have differentiated during the global pandemic as a result of massive virus replication, the very high error rate of reverse transcriptase (RT) and the selective pressure exerted by the immune system. The highly recombinogenic activity of HIV-1 RT has added further complexity to the global diversity of HIV-1 as 43 circulating recombinant forms (CRFs) have already been characterized and a number of unique recombinant forms (URFs) have been identified world-wide [1][2][3]. Most subtypes and CRFs were originally restricted to specific geographical regions or populations, but their distribution is constantly evolving [4]. In order to monitor the evolution of the global pandemic, it is convenient and effective to assign viral clades, which allow evaluation of the local epidemiological trends that result from social changes and migration flows. [6][7][8][9][10][11][12][13]. The recent epidemiology of HIV-1 infection in Western European countries with large immigrant communities has been characterized by increasing genetic diversity and a marked rise in non-B subtype strains among newly diagnosed individuals [14][15][16][17]. It has been assumed that most non-B subtype infections in Western Europe are linked to migration ...

BackgroundTrofile® is the prospectively validated HIV-1 tropism assay. Its use is limited by high costs, long turn-around time, and inability to test patients with very low or undetectable viremia. We aimed at assessing the efficiency of population genotypic assays based on gp120 V3-loop sequencing for the determination of tropism in plasma viral RNA and in whole-blood viral DNA. Contemporary and follow-up plasma and whole-blood samples from patients undergoing tropism testing via the enhanced sensitivity Trofile® (ESTA) were collected. Clinical and clonal geno2pheno[coreceptor] (G2P) models at 10% and at optimised 5.7% false positive rate cutoff were evaluated using viral DNA and RNA samples, compared against each other and ESTA, using Cohen's kappa, phylogenetic analysis, and area under the receiver operating characteristic (AUROC).ResultsBoth clinical and clonal G2P (with different false positive rates) showed good performances in predicting the ESTA outcome (for V3 RNA-based clinical G2P at 10% false positive rate AUROC = 0.83, sensitivity = 90%, specificity = 75%). The rate of agreement between DNA- and RNA-based clinical G2P was fair (kappa = 0.74, p < 0.0001), and DNA-based clinical G2P accurately predicted the plasma ESTA (AUROC = 0.86). Significant differences in the viral populations were detected when comparing inter/intra patient diversity of viral DNA with RNA sequences.ConclusionsPlasma HIV RNA or whole-blood HIV DNA V3-loop sequencing interpreted with clinical G2P is cheap and can be a good surrogate for ESTA. Although there may be differences among viral RNA and DNA populations in the same host, DNA-based G2P may be used as an indication of viral tropism in patients with undetectable plasma viremia.

Parallel analysis of peripheral blood mononuclear cell DNA and plasma RNA from 169 drug-naive human immunodeficiency virus-infected subjects revealed that evaluation of both compartments increases the sensitivity of detection of drug resistance-related mutations, compared with examination of either source alone. Peripheral blood mononuclear cell DNA may play a role in the surveillance of transmitted antiretroviral resistance.

The human immunodeficiency virus type 1 (HIV-1) HR-1 and HR-2 gp41 regions were sequenced in a total of 228 plasma or peripheral blood mononuclear cell samples obtained from an equal number of enfuvirtide-naive subjects for pol genotypic resistance testing in clinical practice. Phylogenetic analysis of the env sequences indicated that 102 belonged to subtype B and 95 to non-B subtypes (31 CRF02_AG, 21 F1, 14 C, 11 A1/A2/A3, 9 CRF01_AE, 9 others) while the remaining 31 were unique recombinant forms. There was considerable variability in the consensus sequence of different clades, particularly in HR-2. The HR-1 amino acid region 36-45, containing all of the enfuvirtide resistance mutations so far characterized, was well conserved except for position 42 where serine and asparagine were unevenly distributed in different subtypes. Enfuvirtide resistance mutations were not present in any sample, reinforcing the expectation that enfuvirtide is effective against many different HIV-1 clades and recombinants. However, some of the mutations outside the amino acid 36-45 region and provisionally suggested to play a role in modulating resistance were detected in a minority of cases. Molecular epidemiological surveys coupled with long-term observation of in vivo response to enfuvirtide and future fusion inhibitors are required to clarify the clinical significance of gp41 natural variability.

Recombination between HIV-1 subtypes B and F has generated several circulating and unique recombinant forms, particularly in Latin American areas. In Italy, subtype B is highly prevalent while subtype F is the most common pure non-B subtype. To investigate the recombination pattern in Italian BF recombinant viruses, we characterized full-length sequences derived from 15 adult patients, mostly Italian and infected by the heterosexual route. One of the BF mosaics was a CRF29, three sequences clustered with low bootstrap values with CRF39, CRF40, and CRF42. With the exception of the CRF29-like sequence, the other recombination patterns were unique, but two possible clusters were identified. Analysis of the gp120 V3 domain suggested a possible link with subtype F from Eastern Europe rather than from Latin America, favoring the hypothesis of local recombination between clade B and F viruses over that of import of BF recombinants from Latin America. HIV-1 subtypes B and F appear prone to generation of unique recombinants in Italy, warranting epidemiological surveillance and investigation of a possible clinical significance.H uman immunodeficiency virus type 1 (HIV-1) is characterized by an extremely broad genetic diversity, caused by high mutation and recombination rates. Currently, HIV-1 group M (''Major''), which is responsible of the worldwide pandemic, is divided into pure subtypes A-D, F-H, J, and K (with subtypes A and F further subdivided into subsubtypes A1-A4 and F1-F2, respectively) as well as 48 circulating recombinant forms (CRFs) of varying epidemiological significance, and untold numbers of unique recombinants that are not known to have spread to many patients. Subtype B has long been predominant in Western European countries but the prevalence of non-B strains has been increasing significantly in past years as a consequence of recent migration waves from Africa, Eastern Europe, and South America.1 Continuous surveillance of HIV-1 molecular epidemiology plays a critical role in the understanding of genetic diversity of HIV-1 and for research purposes such as vaccine development. In clinical practice, pol-based subtype assignment is usually accomplished as a by-product of genotypic antiretroviral resistance testing.A homebrew HIV-1 genotyping assay has been established and offered as a public health service at the HIV Monitoring Laboratory (HML), Department of Molecular Biology, University of Siena, Italy since 1995. 2 The laboratory has been serving a number of clinics for analysis of drug resistance mutations accumulating around 10,000 protease and reverse transcriptase sequences from more than 4000 patients. In addition, some hundred partial gag and env sequences have been obtained for research studies. A recent survey on the whole HIV-1 sequence database at the HML revealed 85% of subtype B sequences and 15% of non-B subtypes, mainly CRF02_AG, F1, C, and A1, based on the pol region.3 In some cases, assignments of subtype based on the pol and env regions were not in agreement. 4 These discrepan...