We also investigated whether the CRT determined in these patients was associated with the stage of disease at the time of presentation. No association was found when considering the whole group of the late presenters included in the study, as shown in Table 2.
An analysis of other factors such as age, sex, infecting virus subtype, route of transmission and IRIS events and their association with CRT is also summarized in Table 2. A statistically significant correlation was found only for the mode of transmission, showing that the intravenous drug users in this study harbor only X4/DM strains.
We then investigated whether X4 or D/M tropism is more frequently observed in patients with very late presentation, defined as a CD4 cell count of less than 50 cells/μl at the time of diagnosis. However, there was no significant correlation found between very late presentation and the tropism of the infecting virus strain (P = 0.27).
Finally, we analyzed whether there is an association between the clinical and laboratory parameters described above and CRT in patients with a very late presentation (CD4 cell counts of <50 cells/μl at first diagnosis of HIV infection). It was shown that all of the very late-presenting patients infected by R5 virus strains (n = 11) were in Centers for Disease Control and Prevention (CDC) stage C3, whereas of those infected with X4-tropic or D/M-tropic virus, only five patients (50%) were in CDC stage C3, one was in stage B3 and four were in stage A3 (P = 0.04). For a more detailed description of very late presenters' CDC stages and presenting symptoms, see Table 3.
Late diagnosis of HIV infection occurs in a substantial proportion of patients newly diagnosed as HIV-positive [20,29], and these represent up to 43% of all newly detected HIV infections in some countries. Treatment of this patient collective poses a challenge to the physician because complications are common and mortality is high . So far, however, there has been little specific data available on the CRT of the HIV strains emerging in patients with late diagnosis.
In our study, in the majority (62%) of ART-naive patients with a late diagnosis, R5 virus strains were detected. Direct comparison of these data with those from other studies is limited by differences in study design, patient characteristics and the tropism assay used. Previous studies [17,30] were performed in chronically infected ART-naive patients but included patients with higher CD4 cell counts than in our population, marking an overall less progressed stage of HIV infection of the cohorts. In these less progressed cohorts, X4 and D/M virus strains were detected at a lower percentage (18%, as predicted using the ViroLogic PhenoSense assay  and 25%, as predicted using the Support Vector Machine (SVM) method ) than in our patient group. This is not surprising, as X4 virus is more likely to be detected in more advanced disease . Shepherd et al.  showed in a study on treatment-naive patients, which also included patients with an AIDS-defining illness, that 52% of the patients harbored X4 virus strains. These data were generated by using the phenotypic Trofile assay for tropism prediction, and the higher percentage found compared with the current study is probably due to differences in test system, study design and population.
The role of ART in the selection of X4 virus variants is controversial. Although some authors have observed an R5-to-X4 CRT switch during ART , others have described a lack of apparent impact of ART on the HIV tropism switch [28,33,34] as well as a relatively high degree of V3 sequence conservation during long-term ART , and there are, therefore, conflicting views on this subject [18,34]. The frequency of X4 and D/M tropism of 38% found in our current study for ART-naive late presenters was similar to that described for late-stage patients who had been heavily pretreated with ART. Studies [6,7] on a collective of heavily pretreated, triple-class drug-resistant patients in late-stage infection showed that 39% of the patients carried X4-tropic and D/M-tropic strains. This comparison of the tropism data between treated and naive patients at a late stage of infection now supports the view that ART itself has a limited role in the switch to X4 tropism. It seems that the late stage and progression of infection itself as well as the immunological impairment that it causes may be more likely to influence the increased appearance of X4-tropic viruses.
The CRT of the infecting strain is believed to be associated with the patient's clinical presentation, as patients infected with X4-tropic virus show more rapid progression to AIDS and death [39,40]. The clinical presentation of the 50 late-presenting patients included in this study was similar in both CRT groups, when applying the CDC staging criteria. However, when only the very late presenters (CD4+ T-cell count <50 cells/μl) were considered, a significant difference in the patients' CDC stage at presentation could be observed. All of these patients infected with R5 virus strains presented with an AIDS-defining disease, whereas, in contrast, 40% of the patients infected with X4 virus were asymptomatic at first presentation (disease stage CDC A3). Earlier studies [41,42] using phenotypic assays show similar results. This finding is difficult to explain. It remains to be determined whether it is associated with a different pattern of immune cell destruction induced by virus strains of different tropism, which may play a significant role in this very late stage of infection.
In this study, the route of transmission via intravenous drug use was significantly associated with the presence of an X4 or D/M virus population. This is not surprising because in contrast to the selective disadvantage that X4 virus has in transmission via the sexual route, as has been described previously (reviewed in ), X4 virus can be transmitted efficiently via intravenous drug use .
A general limitation of studies using genotypic prediction systems is the possible misclassification of R5 virus as X4 and vice versa. According to the recent findings of Bozek et al. , who describe a greater distance in sequence space of V3 sequences in patients with CD4+ T-cell counts below 200 cells/μl, some sequences derived from our patient collective could possibly be mispredicted by bioinformatic methods due to an atypical location of their phenotype in sequence space. However, in that study, misclassification was possible for R5 and X4 virus strains to an equal degree, so in the current study, the overall impact on predicted CRT distribution should be relatively small. Genotypic CRT prediction methods other than geno2pheno are available (e.g. WebPSSM, WetCat; for a comparison, see ) and might yield different results when applied to the data set. We chose geno2pheno for this study because it features an adjustable cutoff, and it can infer CRT in all HIV-1 genotypes. In studies comparing different algorithms for CRT prediction, it achieved a high sensitivity while maintaining a reasonable level of specificity [10,46].
Another possible limitation is the sensitivity of the population sequencing-based approach, which could miss minority X4 variants present in a sample, which are below the detection limit. Furthermore, studies show that the V3 loop is not the only determinant of tropism (for review, see ) and, therefore, tropism predictions made on V3 data alone might not give an entirely accurate picture of the actual viral tropism.
There are no specific guidelines or recommendations for optimal ART regimes in late presenters, and many factors need to be evaluated and considered before starting therapy . The first CCR5 antagonist, maraviroc, has been approved so far only for treatment of therapy-experienced patients infected with CCR5-tropic HIV-1 and receiving OBT . Because of the fact that most virus strains at the time of infection are exclusively CCR5 tropic [17,30], patients in an early stage of HIV infection might benefit the most from receiving maraviroc. Studies [49,50] have, therefore, been conducted to investigate this subject, and recent publications have shown that the prospect of using maraviroc in treatment-naive patients warrants further consideration , considering its pharmacokinetic properties and safety profile. No data have been published so far for maraviroc therapy in antiretroviral-naive late presenters. These patients would possibly gain an additional benefit from a therapeutic regime containing CCR5 antagonists because preliminary data suggest that maraviroc therapy may increase CD4+ T cells from baseline levels, regardless of whether or not the viral load is suppressed , possibly as a result of blocking CD4+ T-cell apoptosis induced by gp120 .
In summary, in our study, in almost two-thirds of ART-naive late presenters, only R5 strains were detectable. Therefore, treatment with CCR5 antagonists in ART-naive patients, even if newly diagnosed at a late stage of HIV infection, should be of value and needs further evaluation.
The authors thank Thomas Urbanek for invaluable technical assistance.
Sample analysis, data correlation, and writing of the article were done by B.S.
Acquisition of samples and clinical data, and article revision were done by K.G.-P., A.R., M.S. and B.S.
Study conception and design, study supervision and article revision were done by E.P.
There are no conflicts of interest.
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