Virological outcome among HIV infected patients transferred from pediatric care to adult units in Madrid, Spain (1997–2017)

The aim of this transversal study was to describe the virological and immunological features of HIV-infected youths transferred from pediatric to adult care units since 1997 vs. the non-transferred patients from the Madrid Cohort of HIV-infected children and adolescents in Spain. We included 106 non-transferred and 184 transferred patients under clinical follow-up in 17 public hospitals in Madrid by the end of December 2017. Virological and immunological outcomes were compared in transferred vs. non-transferred patients. ART drug resistance mutations and HIV-variants were analyzed in all subjects with available resistance pol genotypes and/or genotypic resistance profiles. Among the study cohort, 133 (72.3%) of 184 transferred and 75 (70.7%) of 106 non-transferred patients had available resistance genotypes. Most (88.9%) of transferred had ART experience at sampling. A third (33.3%) had had a triple-class experience. Acquired drug resistance (ADR) prevalence was significantly higher in pretreated transferred than non-transferred patients (71.8% vs. 44%; p = 0.0009), mainly to NRTI (72.8% vs. 31.1%; p < 0.0001) and PI (29.1% vs. 12%; p = 0.0262). HIV-1 non-B variants were less frequent in transferred vs. non-transferred (6.9% vs. 32%; p < 0.0001). In conclusion, the frequent resistant genotypes found in transferred youths justifies the reinforcement of HIV resistance monitoring after the transition to avoid future therapeutic failures.


Scientific RepoRtS
| (2020) 10:16891 | https://doi.org/10.1038/s41598-020-70861-x www.nature.com/scientificreports/ units, have higher mortality 5 and virological failure rates compared to younger children and adults 6,7 . Thus, it is especially necessary to check clinical and virological status of this population, including periodic surveillance studies monitoring the drug resistance mutations (DRM) prevalence to the main ARV families in clinical use in order to ensure proper treatment 8 .
To date, few studies have investigated the clinical status and epidemiological data of transferred patients from pediatric to adult care. Among high-income countries, Spain has one of the most studied and well reported perinatal HIV cohort, with 1,335 HIV-infected children, adolescents and youths registered since 1995 9,10 . The present study updates the demographic, epidemiological and virological features by December 2017 in HIV-1 infected adolescents/youths transferred to adult units in Madrid with available resistance genotypes vs. patients under pediatric care.
Among them, 106 remained in pediatrics care units and 184 were transferred to adult care units from 1997 to December 2017. Table 1 summarizes their demographic characteristics. Both groups were mainly perinatally HIV-infected and 57% of them were female. By December of 2017 the mean age of the cohort was 27 (SD 4.2) years old for transferred and 15.6 (SD 5.7) years old for non-transferred patients. The median age at diagnosis was 1.3 (IQR 0.4-4.6) years for transferred and 0.6 (IQR 0.2-4.6) years for non-transferred patients. Most (90.7%) of transferred were diagnosed before the year 2000 and 74.4% in the 1990s. The transition of patients from pediatric units to adult health care occurred at median age of 18.7 (IQR 17.6-20.8) years old, and mainly (91.8%) after year 2002. The rate of transferred subjects with native Spaniard origin was significantly higher than in the nontransferred (92.9% vs. 68.9%; p < 0.0001). Only 6% of transferred youths were born in Africa or Latin America vs. 29.2% of non-transferred patients (Table 1). Considering the whole study cohort with available resistance data (n = 208), the rate of treated patients was significantly higher than those ARV-naïve (81.1% vs. 18 (Fig. 1b). Figure 2 shows the predicted resistance level to 20 ARV of the most used drug families (NRTI, NNRTI, PI) among the 162 pretreated subjects under study carrying ADR (49 non-transferred and 113 transferred) with available pol sequences. The transferred cohort reported a significantly higher rate of patients with high resistance level to a NRTI family drugs than non-transferred (60.1% vs. 27.2%; p = 0.0081), mainly to d4T (38.2% vs. 11.4%; p = 0.001), AZT (37.3% vs. 11.4%; p = 0.002), ddI (33.3% vs. 11.4%; p = 0.006) and ABC (32.4% vs. 11.4%; p = 0.008). The rate of non-transferred and transferred patients with predicted high resistance level to NNRTI and PI did not show significant differences, except for nelfinavir (NFV), with higher rates of resistance among transferred youths.   Supplementary Fig. S1. We identified the specific drugs with the highest susceptibility in both cohorts, representing interesting alternatives for rescue ART regimens if required. Most PI and the new NNRTI (DOR, RPV, and ETR) were the drugs showing the highest susceptibilities in transferred youths. Nevertheless, subtype B pol prevalence was significantly higher among transferred vs. pediatric cohort (93.1% vs. 68%; p < 0.0001). Non-transferred patients presented a higher prevalence of pure non-B variants at pol (10.7% vs. 3.1%; p = 0.033) and inter-subtype recombinant (CRF and URF) variants than transferred (21.3% vs. 3.9%; p = 0.0001).

Discussion
Transition to adult care is crucial for HIV-infected adolescents. This population faces with important challenges to ensure long-term virological suppression when reaching adulthood. However, little is known about their current health status in each country, despite an expected increase in the number of children being transferred into adult units in the coming years. Several studies have assessed the current state of adolescent survivors of perinatally or early acquired HIV 9, 11-17 . Table 3 shows all related studies on HIV-1 patients transferred from paediatric to adult units worldwide.
During the early nineties, Spain had the highest incidence of mother-to-child transmission in Western Europe among heroin users HIV-infected women, leading to high HIV transmissions in children born between 1980 and 1990 18 . The Madrid cohort is one of the best characterized perinatal cohort in Europe and worldwide, along with the UK/Ireland 11 , the Netherlands 13 and New York City 15 cohorts (Table 3). Future transitioning programs www.nature.com/scientificreports/ will represent a challenge mainly in low-income countries 28,29 where most HIV-infected-children and adolescents live 19, 31 . By the end of December 2017, two-thirds of the perinatally infected patients in our cohort in Madrid (Spain) had reached adolescence and transitioned to adult care. Here, despite similar median age at transfer, transferred youths in the present study were younger at HIV diagnosis (1.3 vs. 2 years old) and at first ART experience (3.4 vs. 5.6 years old) than the same cohort 6 years before 9 . Additionally, over six years, our perinatal cohort had improved their immunological status significantly regarding the rate of transferred achieving CD4 T cells > 500 cells/mm 3 counts (74% vs. 55.3% reported in 2011; p = 0.0031), reaching rates higher than in comparable studies in UK/ Ireland 11 (42%), New York 15 (38.9%) and Argentina 17 (36.3%). The good recovery of CD4 counts in the Madrid cohort could be due to an early diagnosis and treatment and improved ART regimens, in agreement with other studies reporting that better initial status is associated with improved immune recovery [20][21][22] .
Regarding virological outcome, our updated data showed a 27% increase in transferred youths with available sequence with undetectable viral load (UVL) in our Spanish cohort from 2011 to 2017 (38.4% vs. 65.4%; p < 0.0001) ( Table 3). Swedish and Italian cohorts presented higher rates of transferred patients achieving UVL 12,14 and Canadian and Argentina transferred cohorts the lowest 16,17 (42.2% vs. 45%, respectively), despite being considered high-income countries. Nevertheless, in some high-income countries, transferred young people still have high rates of virological failure immediately before, during, and shortly after transition (36% in the Netherlands) 13 , as well as a loss of follow-up after transition (nearly 14% in Spain 23 and in the Netherlands 13 ), mainly in the first year after transfer.
By the end of December 2017, a third of transferred youths still had incomplete viral suppression, and lower median CD4/CD8 ratio than non-transferred patients, a predictor of increased immunoactivation and immunosenescence despite ART 24 . The incomplete viraemia suppression could be explained by partial adherence to treatment, a key problem in adolescence. Moreover, most transferred youths of the study cohort was infected in the mono and bi-therapy era, receiving several suboptimal treatments and selecting a high rate of historic DRM, one of the major obstacles for an effective ART 25 . The complex clinical management in perinatally-infected youths impacts in the current immune-virological control of HIV infection compared to adults and to patients under pediatric care, who probably have received optimal ART regimens. Thus, better immune-virological situation during transition to adult units is expected in future transferred cohorts.
We observed a high TDR rate in the study cohort, mainly in the transferred group. The higher prevalence of resistance found in transferred vs. non transferred individuals could be due to the older age and longer therapy experience with less efficacious antiretroviral treatments and many regimen switches vs. non-transferred. The transferred adolescents had to face the monotherapy and dual therapy regimens available at the time, thus increasing the risk of virological failures and unsuppressed viraemia due to resistance development. In our study, the 4 transferred with TDR were vertically HIV-1 infected adolescents, collective found to have higher risk of treatment failure than newly HIV infected youth, probably as a result of their lifelong infection and their Table 3. Comparison of published studies from HIV-1 patients transferred from pediatric care to adult units worldwide. HIV human immunodeficiency virus, ref reference, No. number of transferred participants in each study, DX diagnosis, cART combination antiretroviral therapy, ART antiretroviral therapy, seq sequences, DRM drug resistance mutation, NRTI nucleoside reverse-transcriptase inhibitor, NNRTI non-nucleoside reversetranscriptase inhibitor, PI protease inhibitor, UVL undetectable viral load ≤ 50 RNA copies/ml at last available viraemia, except from USA study 23 (< 400 cp/ml), cp copies; dash: not provided data. HIV non-B variants include HIV-1 subtypes different than subtype B and recombinants. a DRM found to NRTI + NNRTI, NRTI + PI and NNRTI + PI drug class families. b DRM found to NRTI + NNRTI + PI drug class families. www.nature.com/scientificreports/ heavily ART exposition 6 . Moreover, HIV infected patients during childhood in our transferred cohort were mainly infected during the early 1990s, when Spain had one of the highest rates of AIDS in Europe. The inadequate ART regimens in their HIV-infected mothers could also contribute to the high resistant level found in perinatally infected transferred group. The ART expansion in low-income countries where most pediatric infections occur and the insufficient adherence support, frequent suboptimal ART regimens in HIV-infected mothers and children, lack of routine viral load (VL) and resistance monitoring in most of these settings, can lead to the spreading of resistant viruses among new infections in naïve and treated children. In fact, nearly 85% of naïve non-transferred patients in our study were born abroad or from HIV-infected parents coming from low-income countries, where ART has been expanding in the last years, without the implementation or availability of optimal ART regimens 26 . TDR rate in perinatally HIV-1 infected patients in Madrid was higher than in perinatal cohorts from UK/Ireland (6%) 11,27 , and in most pediatric cohorts worldwide 28 , as well as in adults from Europe (8.3%) 29 and in the Spanish AIDS Research Network of adults (7.9%) 30 . The presence of TDR has important clinical consequences due to the influence of baseline drug resistance patterns in the outcome of first-line ART in children 31 and adults 29 and is a strong predictor of treatment failure.
ADR prevalence among ART-experienced patients has decreased over time in the Madrid cohort for all drug families (Supplementary Table S2). The significant reduction in the rate of non-transferred patients with ADR to NRTI class (from 62.1% in 2011 to 28% in 2017; p < 0.0001) could likely be due to the implementation of LPV/r as a first-line combined antiretroviral treatment (cART) in Spain since 2008, and the withdrawal of NFV in 2007 32 . The significant decrease for ADR to PI in patients under pediatric care and transferred youths in Spain could reflect the improvements in ART due to availability of new drug classes in the last years. Nevertheless, in 2017 transferred patients still maintained the highest ADR prevalence to NRTI (64.1%), since it was the first available drug class for clinical use, ABC and AZT being the most compromised drugs, along with ddI and d4T no-longer-used ARV comparing to non-transferred patients (Fig. 2). This was due to the higher presence of D67N, M41L and T215Y resistance mutations in RT in 42.7%, 38.8% and 30.1% of transferred youth, respectively.
Triple-class resistance was detected in 15.2% of transferred youths, a lower rate than the one previously reported in the same study cohort (17.3%), as in other transferred cohorts in Canada (31.6%) 16 and Argentina (45%) 17 , and higher than in UK (12%) 11 . All patients carrying triple-class resistance in our cohort were born between 1987 and 1996, and 43.9% of them had experienced mono/dual NRTI therapies before cART implementation, which may have led to treatment failure and subsequent ADRM selection due to the incomplete viral suppression 33 . It is important to highlight that the comparison between transferred and non-transferred patients was completely related with the time-period when they were infected and treated, suggesting that a direct comparison may not be accurate in this study.
Despite high ADR rate to the three main ARV families among transferred, our data showed that some NNRTI (DOR, ETR, and RPV) and PI (DRV and TPV) remained good options to rescue the highly pretreated patients in the study cohort. Moreover, nowadays young people could benefit from newly licensed drugs to treat HIV-1 in adults, like cell-entry and integrase inhibitors 34 . Surveillance of TDR and ADR prevalence among HIV-1 infected children and adolescents is critically important in determining if changes to empiric first, second and third-line ART regimens are required 35 .
Regarding HIV infecting variants, infections with non-B variants in non-transferred patients increased significantly from 2011 to 2017 (11.5% to 32%; p = 0.0004), mostly due to the increment of children infected by CRF (6.9% vs. 20%; p = 0.0065). Despite that fact that subtype B was the prevalent variant in the transferred cohort, non-B infections also increased among transferred from 1.9% (2011) to 6.9% (2017) ( Table 3). Inevitably, this viral heterogeneity could affect the efficacy of HIV-1 monitoring, affecting the clinical management of HIV-1 infection or disease progression 36,37 .
The main limitation of the study is that resistance results derived from available pol sequence or resistance profiles closest to December 2017, ranging from 1993 to 2017 but mainly dating from 2005 to 2010 (Table 2). Therefore, resistance patterns may not precisely reflect features in December 2017. Moreover, we only used data from patients with available resistance testing, excluding of the virological study patients without pol sequences. VL quantification assays with different limit of detections differed across patients and years during the clinical follow-up of the study cohort.
The world is home to more young people (ages 10-24 years old) now than at any other time in history, and we need to focus and care for this collective if we want to end the AIDS epidemic by 2030. Our study demonstrated that good clinical management could achieve the goal that most HIV-1-infected patients transferred from pediatric care to adult units may maintain virological suppression and high CD4 counts, decreasing ADR prevalence and improving their clinical status. This highlights the importance of VL and drug resistance monitoring worldwide in all HIV-infected-pediatric and young population for ART optimization if required during the chronic clinical follow-up of infection. www.nature.com/scientificreports/ new pol genotypes recovered from hospitals. We also collected retrospective epidemiological-virological data from clinical records closest to December 2017: origin, gender, age, HIV transmission route, HIV diagnosis date, antiretroviral therapy (ART) experience, CD4 and CD8 counts (percentage and cells/mm 3 ), CD4/CD8 rate, and viral load (HIV-1 RNA copies/ml of plasma, cp/ml). This study was approved by the Clinical Research Ethical Committee at University Hospital Ramón y Cajal (Madrid, Spain). All methods were carried out in accordance with relevant guidelines and regulations. Informed consent was obtained from all subjects or, if subjects are under 18, from a parent and/or legal guardian.

Study population.
Drug resistance analysis. The acquired HIV drug resistance mutations (ADR) in pretreated patients to nucleoside reverse-transcriptase inhibitors (NRTIs), non-nucleoside reverse-transcriptase inhibitors (NNRTIs) and major protease inhibitors (PI) were defined by the HIVdb Program Genotypic Resistance Interpretation Algorithm v8.9-1 (Stanford University, Palo Alto, CA, USA) 38 . In drug-naïve patients, the prevalence of transmitted drug resistance mutations (TDR) was established according to the mutation list as recommended by the WHO 39

Statistical analysis.
To compare the pediatric and transferred cohorts the Fisher exact test and Chi-square test were used for categorical variables. The unpaired Student t test or the Mann-Whitney test was performed for continuous variables. Means and standard deviations (SD) were used for normally distributed data, and medians and interquartile ranges (IQR) for data that are not normally distributed. To compute the 95% confidence interval (95% CI) we use the hybrid Wilson/Brown method for the sensitivity/specificity and the Newcombe/Wilson method to calculate the difference between proportions. All analyses were performed by using GraphPad Prism 8.0.1. Two-sided p-values of < 0.05 were considered statistically significant.