This study investigates the combined effect of selected parameters and the CCR5 Δ32
allele on long term survival among HIV-1 infected patients in the era of cART. An array of factors influencing survival were analyzed in this study including demographics as well as clinical and immunological parameters. The frequency of the CCR5 Δ32/wt
heterozygotes in this study was 14.0% and is consistent to the previous reports for Caucasian populations, while use of archived samples allowed to avoid the bias related to the survival benefit of the mutation in HIV infected patients who may have died 
. The near-significant difference between the male and female gender frequency might be related to the transmission route – higher number of women was infected sexually. It is possible that the risk of HIV transmission is differently modified by the Δ32
allele for every gender, depending on the type of exposure.
An association between the Δ32
allele and delayed progression to AIDS has previously been reported including a suggestion that this deletion is the strongest protective factor for both viral and immununologic control of the HIV-associated disease and improved survival outside the HLA system 
. In the study by Hendrickson et al. 
, both time to viral suppression and AIDS free survival following cART initiation was significantly longer in the group with the Δ32
allele. In the meta-analysis by Ioannidis et al., the relative hazard for both AIDS and death was notably lower among the CCR5 Δ32/wt
heterozygotes if compared to the individuals homozygous for the wild-type variant 
. The beneficial effect of the CCR5 Δ32/wt
genotype was also found by Brumme et al. 
, who observed improved survival in the median >5 year period after starting the antiretroviral therapy in the univariate analysis adjusted for baseline age, lymphocyte CD4 count, viral load and AIDS defining conditions.
In our study the number of AIDS-free individuals with the CCR5 Δ32
variant was significantly higher at the time of HIV diagnosis, however for the entire follow-up period the number of AIDS cases was not notably different between the groups with and without this allele. This could be related to the fact that the cohort used for this analysis consisted of a large number of late diagnosed individuals, in 372 (73.4%) cases infection was diagnosed in either the symptomatic stage or with baseline CD4 count<350 cells/µl, which is characteristic for HIV infections diagnosed in Poland 
. The protective effect of this deletion may have been lost due to the time-dependency of the beneficial effect, as reported before 
. Similarly, the slightly higher prevalence of the Δ32
allele among antiretroviral naive patients is probably related to a higher number of patients asymptomatic at baseline who would not require cART. Other differences of borderline statistical significance for the CCR5 Δ32
allele frequency include a lower frequency among women, confirming the finding by Philpott et al., of a more extensive protective effect of the CCR5 Δ32/wt
heterozygous genotype in women 
while a lower prevalence of the heterozygotes among heterosexually infected patients might be related to the fact that high CCR5 levels are required for these primarily macrophage-tropic sexual infections 
In the univariate analysis of all the factors significantly modifying all-cause mortality the strongest protective effect was observed for cART as well as CD4 metrics, namely nadir, zenith lymphocyte CD4, time with CD4 count above 500 cells/µl and AIDS-free observation. Effects of these parameters were extensively studied before and remain well documented 
. These influences might be additionally modified by the Δ32
allele, found also to be a statistically significant protective influence on mortality, as it has been reported that in individuals bearing this variant time of progression to AIDS after initiation of cART as well as time from AIDS diagnosis to death is increased 
. In our study the Δ32
allele was associated with decreased mortality risk observed for as long as 15 years of follow-up. This association remained significant after adjustment for other factors strongly influencing mortality: gender, nadir and zenith CD4 count, time with CD4 >500 cells/µl, most recent CD4 levels and AIDS diagnosis.
It must be noted, that the CCR5 Δ32/wt
genotype was associated with better survival in untreated individuals only, which is probably associated to the slower development of the symptomatic disease as suggested by Brumme et al., 
while among cART treated individuals the correlation with better survival was insignificant, however this analysis was performed on smaller number of patients (55 with the CCR5 Δ32/wt
genotype with 23 on cART for 5 years and 6 for more than 10 years). No significant effect of the Δ32
allele, survival and AIDS free survival was also found by Laurichesse et al. 
. Moreover, Brumme et al., did not find the association between the CCR5 Δ32/wt
genotype and time to virological failure in the study of 436 individuals observed for the median follow-up of 22 months 
. Similarly, Bratt et al, in the study of 147 patients, which included similar (37%) percentage of AIDS diagnosed patients as our analysis, observed little influence of the CCR5 Δ32/wt
genotype on virologic treatment efficacy 
. Lack of beneficial effect of the Δ32
allele among antiretroviral treated patients observed in our study might be associated not only to the size of the group, but also differences in the time from the infection to treatment initiation, which is related to late diagnosis of HIV and high prevalence of AIDS at the time of diagnosis. However, it must be noted that the Δ32
allele has been associated with the decrease in the likelihood of viral suppression failures, defined as HIV-RNA ≥200 copies/ml at 16–28 weeks of therapy, accelerated viral suppression to <200 HIV-RNA copies/ml, as well as significantly better virologic response to antiretrovirals both at 6 and 12 months following treatment initiation 
. Moreover, a higher rate of the sustained virologic suppression during the observation period of up to 5 years of antiretroviral treatment in the CCR5 Δ32
heterozygous patients was noted 
In adjusted multivariate analyses both AIDS diagnosis and antiretroviral treatment remained significant predictors of mortality. Moreover, it was confirmed that of all the CD4 metrics studied, the latest CD4 count is the strongest predictor of mortality, as has been suggested before 
. When the time-dependency for the six factors included into the multivariate model was studied, a continuous significant positive effect, regardless the period of observation both for this variable and history of cART, was observed. This confirms the value of these parameters and emphasizes the need for early introduction of the antiretroviral treatment. Stable CD4 count of >500 cells/µl for at least 180 days became a significant parameter after 30 months of observation, which is probably related to the time dependent immune recovery. Interestingly, the continuous protective effect of the CCR5 Δ32/wt
genotype was observed over 36 months of follow-up with stable HR of 2.5–2.6 noted up to 96 months, the end of observation period. This suggests that in the cART era the protective effect of this variant on survival is not lost over time as was observed for the Δ32
allele and AIDS diagnosis. Hazard ratio effect plots for gender and diagnosis of AIDS proved time-dependent, with female gender associated with significant protection up to 108 month of observation while for AIDS two periods associated with favorable prognosis in AIDS free patients were noted, namely from months 36 to 54 and 108 to 180 months. The reason for this time dependent relationship remains to be elucidated.
Studies analyzing the impact of the CCR5 Δ32 deletion on long term survival among HIV (+) patients in the cART era remain sparse, especially with implementation of Cox regression models adjusted for clinical variables significantly contributing to the mortality risk.
A potential limitation of the study was use of interval censoring for six cases when the exact date was unknown, with the death date fixed as the median date between the last observation time and information that the patients actually died. Another limitation is related to the lack of possibility to observe the patients from the time of seroconversion, however in the setting of the late testing and referral of majority of cases with symptomatic immunodeficiency such a study was not feasible. Another limitation is related to the lack of information on adherence, therefore the results could not be controlled for this factor.
Host genetics strongly interacts with HIV leading to the modification of the course of infection. Our finding of the beneficial effect of the CCR5 Δ32 on long term survival in conjunction with well established factors influencing mortality provides an insight into the interplay between genetic and virus/host-related factors. Such an effect is of the greatest importance in the late-tested populations, such as the one in Poland, allowing for the longer period of delay from infection to disease detection and treatment initiation, whereas it is of the lesser importance in the cART treated individuals. Testing for the CCR5 Δ32 mutation in clinical practice might be useful for elucidation of the reason for delayed progression of HIV infection, however it should probably not influence decision on antiretroviral treatment initiation or modification.