Baseline Features of the Transferred Population
A total of 112 patients of the Madrid cohort of HIV-infected children transferred from pediatric services to adult units in different hospitals in Madrid between 1997 and December 2011 were selected for this study. Baseline characteristics of the non-transferred (n
131), total transferred (n
112), and transferred patients with available genotypic profile (n
63) are summarized in . All transferred patients were HIV-1 diagnosed at childhood (mean 2 years of age), the majority were born in Spain (91.9%) and mainly infected through MTCT (93.7%). Only a few (12.5%) were adopted. Most (81.3%) were diagnosed along the 1985–1994 period. The median duration of follow-up was 13.2 (Standard Deviation, SD 5.2), 15.6 (SD 4.5), and 16.7 (SD 3.6) years for non-transferred, transferred (n
112) and transferred with available resistance genotypic profile, respectively.
Baseline characteristics of the non-transferred, transferred and transferred with available genotypic profile patients.
Advanced stages of immunosuppression were observed as a result of the long term infection and scarce effective antiretroviral availability before 1996. Over two thirds of transferred patients reached less than 15% CD4+ cell counts and half (57.1%) reached <200 cells/mm3. The mean nadir CD4+ T-cells count was 10%. Monotherapy was the first ARV treatment in 59.8%, mainly with AZT (79.1%), 23.2% started with combined therapy (including AZT backbone in 88.4% of them) and only 14.3% with HAART.
In the transferred cohort compared to the non-transferred, a statistical significant lower number of African patients were found (1.8% vs. 13.7%, p<0.05), a significant higher number of children reached nadir CD4-Tcell values below 200 (57.1 vs. 26.7, p<0.05) and a lower number of patients achieved CD4 T-cells over 500 (9.9% vs. 22.9%, p<0.05). No statistical differences were found in the baseline studied characteristics between transferred patients with and without available genotype ().
Features of the Population at the Time of Transfer
Characteristics of the study population at the time of transfer to adult units are shown in . By the time of transfer, the mean age was 18.9 years and the mean CD4+T-cells count 627.5 cells/ml. 5.4% presented less than 15% CD4+T-cells, 66% more than 25% and 55.3% more than 500 CD4+ cells/ml counts. Nearly all (98.2%) had presented signs and symptoms of AIDS according to CDC classification 
, which were severe in 34.8% of cases. Immunological status at the time of the transfer revealed an immunologically severe suppression in 66.9% (CDC stage 3). Among the 102 patients with available viral load data, 56.2% had ≤500 RNA-copies/ml and 38.4% undetectable viraemia (≤50 RNA-copies/ml).
Characteristics of the non-transferred, transferred and transferred with available genotypic profile patients by December 2011.
Comparison among transferred and non-transferred patients by December 2011 revealed that transferred patients had a worst immunological-virological profile comparing to the non-transferred group. A lower number of transferred patients were categorized in clinical status A (23.2% vs. 37.4%, p<0.05) and achieved undetectable levels of viraemia (38.4% vs. 64.9%, p<0.05). T-cell CD4 counts (either ≥25% or >500 CD4+ T-cells) were also lower among transferred and a lower number of transferred were on HAART (84.8% vs. 94.6%, p<0.05). No statistical differences were found in studied clinical features by December 2011 between transferred patients with and without available genotype ().
ART Experience among Transferred Patients
The transferred cohort started any type of ART with a median age of 5.6 years (SD 3.5 years) and the median duration of ART was 11.5 years (SD 4.8 years). Only 3 (2.7%) of the 112 transferred individuals remained drug naïve at transfer and the rest (97.3%) were ART experienced. Most (84.8%) were receiving HAART, 9.8% had stopped treatment and 2.7% were receiving combined therapy at the transfer time according to clinical reports. The most commonly used HAART combinations in our cohort were 2NRTI+1NNRTI (32.6%) and 2NRTI+1PI (28.4%). The pretreated patients presented a long treatment history and had experienced several different ART combinations; the mean number of regimens was five, with at least 3 HAART regimens in 48% of them. The main ART families were NRTI, PI and NNRTI, 94.6%, 84.8%, and 72.3% respectively (). Triple class experience was found in almost two-thirds of patients. Use of other drug families was scarce and only three transferred patients had experience with the fusion inhibitor, enfuvirtide and one of them also had received raltegravir, an integrase inhibitor. Adherence history was assessed according to clinical charts data but was not available in all cases.
High Prevalence of HIV-1 Resistant Variants in Pretreated Transferred Patients
Among the 112 patients transferred before the end of December 2011, only in 63 (56.2%) subjects drug resistance genotypes could be analyzed. Among them, in 48 (76.2%) cases the pol
sequence had been previously reported by our group 
. In the remaining 15 patients, six patients had an available plasma specimen stored in the HIV-1 Spanish Biobank and new HIV-1 pol
sequences were newly generated as previously reported 
. Other 9 patients presented a genotypic resistance profile derived from pol
sequences obtained from plasma specimens recovered from clinical routine drug-resistance tests performed in hospitals where the patients were or had been under follow-up. However, no fasta format sequences of any profile were available. Clinical specimens in all 63 genotyped samples were obtained before the transfer time to adult units: during 1993–1999 (5 genotypes), 2000–2004 (27 genotypes) and 2005–2010 (31 genotypes). The median age of patients when genotypic data was generated was 14.7 years old (SD 4.4). The 63 patients with available genotypic data included five genotypes (accession numbers HQ426734, HQ426806, HQ426860, HQ426867, HQ426893) performed before treatment was initiated. Out of these five, only 2 patients remained drug naïve until they were transferred to adult units while the other 3 received any ART during their follow-up. None of these sequences harboured TDR mutations.
Among the 63 transferred patients of the Madrid cohort of HIV-infected children with available pol sequences or resistance profiles, the prevalence of HIV drug resistance mutations was analyzed according to the drug class family. Fifty-eight were pretreated and 5 remained drug-naïve at sampling time. No TDR mutations were found among the pol sequences from the 5 transferred naïve subjects. The most prevalent DRM found in the 58 transferred ART-experienced patients were: for PI, L90M (27.5%), V82A (15.7%), M46I (13.7%) and D30N (9.8%); for NRTI, M41L (48.1%), D67N (40.4%), T215Y (40.4%), L210W (34.6%), M184V (25%), T69D (23.1%), and K219Q (23.1%); and for NNRTI, K103N (19.2%), Y181C (7.7%) and G190A (7.7%).
Global resistance prevalence among the 58 transferred ARV-exposed pretreated patients with available pol sequences or resistance profiles was 50.9% for PI, 76.9% for NRTI and 36.5% for NNRTI (). No primary drug resistance mutations were found among naïve subjects. According to our data, no statistical differences were found between the resistance rate and the duration of treatment (data not shown).
Comparison of HIV drug resistance mutations prevalence according to drug class family in the non-transferred and the transferred pediatric cohorts.
Higher Prevalence of Drug Resistance Mutations Among Transferred Patients than in Non-transferred
Besides assessing the global prevalence of DRM in the 58 pretreated patients of the transferred cohort, prevalence of DRM was also compared to the non-transferred cohort. The first included the 58 transferred and pretreated patients with available genotypic data compared to the 131 non-transferred pretreated pediatric patients from the Madrid cohort of HIV-1 infected children (). All 131 non-transferred pediatric patients had available resistance genotype, previously reported 
and with available GenBank accession numbers.
Transferred patients tended to present higher DRM prevalence when compared to non-transferred children (). Drug resistance mutations were found in a higher number of transferred patients than in the non-transferred pretreated population under follow up in Madrid for PI (50.9% vs
. 36.8%, p
NS) and for NRTI, 76.9% vs
. 62.1%, p
NS) and lower for NNRTI (36.5% vs. 40.5%, p
NS). However, resistance prevalence was significantly higher among transferred patients for the PI+NRTI combination (19% vs
. 8.4%, p<0.05). Triple resistance was similar to non-transferred pediatric patients (17.3% vs.
Higher Predicted Level of Resistance to PI and NRTI among Transferred Patients
shows the comparison of the predicted level of resistance to each drug in all pretreated patients from the two study cohorts. Analysis of the genotypic resistance interpretation revealed that transferred adolescents presented a significantly higher predicted level of resistance to all drugs from the PI and NRTI families probably explained by the long-term therapy history of these patients. Half of the transferred patients carried infecting HIV-1 variants resistant to NFV, followed by AZT and d4T (nearly 40% of them). Similar predicted resistance was observed for 3TC and FTC in the non-transferred and transferred study groups (around 20%). Between nearly 20% and 30% of the adolescents in Madrid were infected with variants resistant to the remaining PI drugs, except for TPV/r and DRV/r, the new PI drug generation. EFV and NVP were the NNRTI with the most compromised susceptibility in both cohorts (around 20–30%), being higher in non-transferred patients. Data revealed a low (2–5%) predicted resistance level to etravirine and rilpivirine, the new NNRTI drugs; resistance levels to these drugs remained low in a gap between 2% and 5% for both studied cohorts.
Predicted resistance level to antiretroviral drugs in pretreated patients from the two studied cohorts.
Low Prevalence of HIV-1 Non-B Variants in Transferred Patients
Most (98.1%) of the transferred patients were infected by subtype B, the most prevalent HIV-1 variant in North America and Western Europe, including Spain 
. Only one transferred perinatally infected female carried a “pure” sub-subtype A2. She was a white-Caucasian, vertically infected and born of Spanish parents in 1992. Prevalence of HIV-1 non-B variants among the 54 transferred patients with available pol
sequence was low (1.9%, 1/54), compared to the 11.5% (15 patients) found among the 131 non-transferred children of the same pediatric cohort () or the 12.2% reported n the Spanish cohort of antiretroviral treatment-naïve HIV-infected patients 
. Of interest, recombinant viruses were absent in transferred patients although found in 60% (9/15) of infections caused by non-B variants in the non-transferred pediatric cohort, respectively.
(PR and/or RT) sequences from 48 (88.8%) of the 54 transferred patients included in this study had been previously submitted to GenBank 
: HQ426715, HQ426719, HQ426725, HQ426728, HQ426734, HQ426766, HQ426768, HQ426779, HQ426780, HQ426788, HQ426799, HQ426806, HQ426807, HQ426818, HQ426826, HQ426840, HQ426842, HQ426847, HQ426850, HQ426857, HQ426860, HQ426861, HQ426866, HQ426867, HQ426868, HQ426869, HQ426874, HQ426879, HQ426880, HQ426883, HQ426889, HQ426890, and HQ426893. In reference 
: JQ351951, JQ351960, JQ351984, JQ351986, JQ351988, JQ351989, JQ351995, JQ351997, JQ352005, JQ352006, JQ352010-JQ352012, JQ352014, and JQ352021. The 6 newly generated pol
sequences for this study were submitted to GenBank with the following accession numbers: JQ828989-JQ828994.
GenBank accession numbers for the 131 sequences from non-transferred children were previously reported