Pre-exposure prophylaxis (PrEP) has become a useful tool to reduce the transmission of human immunodeficiency virus (HIV) in key populations. In this article we assessed the effectiveness, safety, adherence, sexually transmitted infections (STIs) dynamics, and frequency of anal dysplasia among a real-life cohort of PrEP users in Northwest Spain.A retrospective cohort study was undertaken in the Alvaro-Cunqueiro Hospital, Vigo which included every individual who started daily emtricitabine/tenofovir-disoproxil-fumarate (FTC/TDF) between November-2019 and October-2021. Clinical and epidemiological data were obtained from the patient's medical records. The effectiveness and safety of FTC/TDF were assessed by HIV serology and renal function monitoring every 3 months. Anal, urethral, and oropharyngeal exudates were collected quarterly after the baseline visit.p = 0.013). Among the study population included in the anal screening programme, the frequency of dysplasia was 9%.A total of 126 individuals were considered eligible, most of the participants had previously been diagnosed with a STI (60.3%), 22% had consumed recreational drugs in the year prior, and 13% had engaged in chemsex. At the end of the follow-up, no cases of HIV infection were detected; 3 patients had discontinued FTC/TDF because of side effects but none of them had presented renal toxicity. In addition, the diagnosis of STIs during the follow-up was common (100 cases in 54 patients). Moreover, engagement in chemsex was more common within this latter group . However, a high number of STIs were diagnosed, especially among patients who engaged in chemsex. In recent years pre-exposure prophylaxis (PrEP) has become a useful strategy to reduce the transmission of human immunodeficiency virus (HIV) . PrEP reWhether FTC/TDF is related to a decline in the estimated glomerular filtration rate (eGFR) remains controversial and so far, contradictory data has been reported in this respect , 5. ImpoNevertheless, in the context of PrEP, some concerns have emerged regarding the incidence of sexually transmitted infections (STIs) other than HIV. In this respect, several studies have reported a high incidence of STIs among PrEP users , a decreIn November 2019, an FTC/TDF based PrEP programme was implemented by the Public Health Service in Northwest Spain . This plan included the prescription of a daily dose of FTC/TDF, kidney function monitoring, and STI screening every 3 months for individuals at a high risk of HIV infection. Herein, we report our experience with a real-life cohort located in Northwest Spain, including data from the time the use of PrEP was initiated in our health area. The PrEP effectiveness, safety, sexual behaviours, drug consumption, and STI incidence during the study period were all reviewed.An observational retrospective cohort study was undertaken by the Infectious Diseases Department in the University Hospital Complex of Vigo which has a catchment area population of 450,000 inhabitants. The medical records of individuals who requested PrEP administration were reviewed. The Galician PrEP programme  started Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) using a Cobas® 6,800 system . Serum samples were tested for HIV , Treponema pallidum , and hepatitis C virus . The Roche Cobas HPV test was used to detect high-risk human papillomavirus (HPV) in anal cytology samples and to genotype HPV-16 and HPV-18 as well as a grouped category of high oncogenic risk HPV . The anal cytology was processed in the Pathology Department according to routine procedures and later categorised based on the standard criteria. Biopsies obtained during the anoscopy were reviewed by an expert pathologist and classified according to the Bethesda criteria.Samples were processed at the Microbiology Service at the University Hospital Complex of Vigo according to laboratory standards. Anal, urethral, and oropharyngeal samples were screened for At the time of their baseline visit all the patients were invited to participate in the Anal Dysplasia Surveillance Programme. For those who accepted the offer, an anal cytology sample was obtained during the anal exam. A high-resolution anoscopy (HRA) was then conducted by an expert surgeon to screen for altered anal exam results or abnormal cytology results .A past STI was defined as any STI diagnosed prior to the inclusion in the PrEP programme; baseline STI status was defined as a venereal disease diagnosed during the baseline visit (prior to FTC/TDF prescription); and finally, an incident STI was defined as a new episode of a STI that occurred during the follow-up after starting PrEP.Instituto de Investigación Sanitaria Galicia Sur. Quantitative variables were expressed as the median and interquartile range. Qualitative variables were shown as absolute values and percentages. Categorical variables were compared using χ-squared tests or the Fischer exact test, as appropriate. Quantitative variables were compared by employing Man–Whitney U-tests. P-values < 0.05 were considered significant in all cases. The statistical analyses were performed using Statistical Package for Social Sciences (SPSS) software .The study data were collected and managed using REDCap electronic data capture tools hosted at the This study was approved by the Ethics Committee of Pontevedra-Vigo-Ourense (reference 2021/311). The need for informed consent was waived because of the retrospective design of this work. The STROBE guidelines were used to ensure the reporting of this study.n = 28) had consumed recreational drugs in the year prior, and 12.7% (n = 16) had consumed recreational drugs during sex (chemsex). Most of patients had previously been diagnosed with an STI, with syphilis being the most frequent .During the study period, a total of 136 individuals requested the prescription of PrEP in the Infectious Disease Unit. Of these, 10 were excluded in the final study cohort, in 6 cases because the patients had been included in a clinical trial, 3 individuals tested positive for HIV-infection, and 1 patient eventually declined to use PrEP. Thus, a total of 126 patients were finally considered eligible for this study. The baseline characteristics of the study population are shown in n = 38) tested positive for a bacterial STI, with CT  and NG  being the pathogens most commonly isolated.According to the local protocols, a baseline STI screening was performed prior to the administration of FTC/TDF. Three patients tested positive for HIV-infection and were therefore excluded from the PrEP programme. An acute HIV-infection was suspected in the first patient (he referred high-grade fever and odynophagia) while the other two individuals were asymptomatic; two were born in Latin America and the other was born in Spain. The ages were 19, 24, and 28 years old, two of them had a previous history of a STI . None of them had previously received post-exposure prophylaxis (PEP). All three patients had tested negative for HIV-infection in the prior year. No HCV infection cases were detected. Among the remaining patients, 30.2% (n = 100) of the participants remained on PrEP, 14% (n = 17) were lost to follow-up, and PrEP was no longer indicated  in 5% (n = 6) of the cases. FTC/TDF was discontinued because of safety or toxicity concerns in 3.2% (n = 3) individuals, 2 of them because of digestive intolerance and 1 as the result of skin toxicity (severe folliculitis attributed to the drugs). No patients stopped FTC/TDF treatment due to kidney toxicity.The median follow-up time was 13 months, and no cases of HIV or HCV infections were detected. At the end of the study period 79% (n = 54) were diagnosed with at least one STI, with NG  and CT  infections accounting for most cases (n = 8) and the majority of cases were of early asymptomatic and latent syphilis (n = 7); only one patient presented a chancre. In addition, genital herpes was detected in n = 7 individuals, while other STIs  were diagnosed in 10 patients.During the follow-up, 43% of patients , especially among those who practiced chemsex . Patients who were diagnosed with an STI at the baseline visit exhibited a higher proportion of incident STIs, although this difference was not statistically significant . Conversely, the age of the first sexual encounter, number of sexual partners, and prior prescription of a post-exposure prophylaxis (PPE) treatment, did not differ between the two groups.Patients were classified into two groups according to whether an incident STI was detected or not; the comparison between these two groups is shown in n = 97) of the participants accepting this offer (n = 22) individuals and HRA was performed in n = 26; 9.3% (n = 9) patients were diagnosed with an anal SIL, with most of them being low-grade (LSIL) except for 1 high-risk (HSIL) case. The prevalence of HPV within this subgroup was common (69.1%), in most of these cases due to pHR-HPV (55.7%), with HPV-16 (17.5%) and HPV-18 (3.1%) diagnoses being less usual. Most patients (86%) had received the nine-valent HPV vaccine prior to their inclusion in the anal dysplasia screening programme.During the baseline visit, all the patients were invited to join an anal squamous intraepithelial lesion (SIL) surveillance programme, with 77% . Real-life studies also provide valuable data concerning the safety and effectiveness of FTC/TDF and STI dynamics after the implementation of PrEP programmes worldwide. To the best of our knowledge, this is the first study to report real-life data after the implementation of a PrEP programme in Northwest Spain. In this cohort, FTC/TDF was highly effective with no HIV infections detected during the study period and no treatment discontinuations because of renal toxicity. The main cause for PrEP discontinuation in our study population was the presentation of adverse gastrointestinal events 2.4%); interestingly, these were also more common in the FTC/TDF arm compared to the placebo  in the IPERGAY clinical trial .4%; inte.In the recent years, access to HIV, STI and PrEP care has become more challenging owed to the COVID-19 pandemic , 18. NevChlamydia trachomatis infection was frequently isolated from anal samples, which has been described as a main risk factor for HIV acquisition , including infection with HPV-16 (22 vs. 18%)  . Of notevs. 18%) .In addition, SIL surveillance programs have been developed for cervix and anal cancer prevention which have led to a decline in the incidence of HPV-driven dysplasia. Anal squamous cell carcinoma is a rare disease among the general population, but its incidence is disproportionally high in HIV-infected MSM . WhetherOf note, chemsex has emerged in recent years as a major concern both among PLWH and PrEP users. Chemsex is associated with the consumption of various psychoactive recreational drugs , group sex, and condomless intercourse that can last hours or even days . The oveChemsex is also associated with harmful effects in terms of mental health, addiction, and an increased risk of STI/HIV acquisition. Depression, anxiety, and poorer mental health scores are all more common among people who practice chemsex . MoreoveMycoplasma genitalium , and CT isolates were not tested for lymphogranuloma venereum Chlamydia trachomatis L1–L3 serovars.Finally, it is important to note that our study had several limitations. Firstly, the retrospective design of the work might have resulted in some data loss. Our sample size was limited, likely because of the COVID-19 lockdown and social restrictions that may have reduced the demand for PrEP regimens. In addition, in our health area, the implementation of PrEP is restricted to daily treatments while on-demand use of FTC/TDF is discouraged, which might dissuade some patients from using it. Secondly, the sexual habits of the participants  were only recorded at the baseline visit, not during the use of PrEP. Thus, any behavioural changes during the follow-up period were not considered in this current work. Thirdly, no renal biomarkers for TDF kidney toxicity  were measured, although no patients stopped using TDF for renal safety reasons. Finally, at our centre, the screening samples were not routinely tested for In our study population, the daily use of FTC/TDF was effective and safe. Most patients continued the follow-up for 1 year and treatment discontinuations due to adverse effects were uncommon. Both the incidence and prevalence of STIs were high, especially among chemsex users. Finally, the presence of HPV-driven dysplasia should not be ignored, and long-term studies will be needed to analyse its relevance among PrEP users.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.This study was approved by the Ethics Committee of Pontevedra-Vigo-Ourense (reference 2021/311). Written informed consent for participation was waived for this study in accordance with the national legislation and the institutional requirements.AP-G: designed data collection tools, monitored data collection, analysed the data, and drafted the manuscript. MR, SR-R, CV-E, GP, and EF: collected data. PC: revised the manuscript. CP and AO: analysed the data and revised the manuscript. EP: designed, analysed, and revised the manuscript. All authors contributed to the article and approved the submitted version.Instituto de Investigación Carlos III (ISCIII) with reference number CM20/00243.AP-G principal investigator is hired under a Río Hortega contract financed by the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


We created a transition readiness score for adolescents with perinatally-acquired HIV as they transition from pediatric to adult care. Of the 199 adolescents who transitioned to adult care, 84 (42%) had viral suppression (< 200 copies/ml) one year after transition. Adolescents on first-line ART, with documented HIV status disclosure, and higher rating on the HIV Adolescent Readiness to Transition Scale had significantly higher odds of viral suppression after transition. Conversely, females, those with older age at ART initiation, and those with prior alcohol use had significantly lower odds of viral suppression after transition. Using these data, we created a transition readiness score organized into low, intermediate, and high levels of transition readiness. This transition readiness score can be used to identify adolescents who are likely ready to transition to adult care and identify additional areas for intervention to improve the likelihood of successful transition for those with lower transition readiness scores. South Africa has an estimated 330,000 adolescents living with perinatally-acquired HIV, the highest of any country in the world , 2. As tIn South Africa, retention in care among adolescents living with HIV who transition to adult care is suboptimal . This trIn other settings, transition readiness assessments are used for adolescents with chronic medical conditions such as sickle cell disease, cystic fibrosis, childhood cancer, and chronic renal disease to assist with timing of transition to adult care –16. ThesTo address this gap in adolescent HIV care, we identified factors associated with successful transition from pediatric to adult care for adolescents living with HIV in South Africa. We then used these factors to create a transition readiness score to assist clinicians in determining readiness and identifying modifiable factors to prepare for transition.Prince Mshiyeni Hospital is a regional/district hospital located in the township of Umlazi outside of Durban, South Africa. The outpatient pediatric HIV clinic provides treatment for > 800 children and adolescents living with HIV— all of whom will require transition to adult care. In this setting, transition to adult care typically occurs after an adolescent’s 12th birthday but may occur later based on clinician judgement. Adolescents initially transition to the adult clinic at Prince Mshiyeni Hospital before being transferred to local clinics at a later date.We recruited study participants from the pediatric clinic of Prince Mshiyeni Hospital during their routine clinic visits between August 2017 and May 2019. We offered enrollment to a convenience sample of consecutive adolescents (age > 12 years old) living with perinatally-acquired HIV who were transitioning from pediatric to adult care. To avoid accidental disclosure of HIV status by participating in the study, adolescents were referred to the research team by their primary pediatrician after they felt that the adolescent sufficiently understood their HIV status. To assess transition readiness at the point of transition, we enrolled participants on their last day in the pediatric clinic prior to transitioning to the adult clinic. In this exploratory study, we anticipated that a sample size of 200 participants and viral suppression rates of approximately 50% after transition would give sufficient power to evaluate up to ten independent variables using the heuristic rule of ten events for every independent variable. Adolescents with developmental delay interfering with the consent procedures were excluded from participation. Adolescents < 18 years old assented to study participation and written consent was obtained from the primary caregiver. Adolescents 18 years or older provided their own informed consent.At enrollment the participants provided basic demographic information, completed a questionnaire and additional data was extracted from the medical record. The questionnaire included drug and alcohol use (Youth Risk Behavior Survey) , social We first performed bivariable analysis using logistic regression models assessing viral suppression adjusting for potential covariates including: age at ART initiation, ART regimen, biological sex, HIV disclosure status , drug use, alcohol use, social support, self-esteem, and transition readiness . We then performed a multivariable analysis evaluating viral suppression using multivariable logistic regression with Huber-White robust standard errors, based on the continuous total value of the HARTS and covariates with a p-value of < 0.2 on bivariate analysis. In the multivariable regression, we also assessed for interaction terms among the covariates and retained any interaction terms whose p-value was < 0.05. All statistical analyses were performed using Stata 13 .REF)). For each categorical covariate, the lowest category was considered as the reference and the midpoint as the reference value. We then set a base constant (Bconstant) to reflect one point in the point scoring system. For our base constant, we utilized the smallest coefficient  considering the increase in viral suppression associated with a 10-point increase in HARTS . The score for each category of each variable was computed as β(W-WREF)/ Bconstant rounded to the nearest integer. A unit score therefore represents the increase in the likelihood of viral suppression associated with a 10-point increase in the HARTS. Finally, categories were determined according to the range (minimum to maximum) of the total score. Tertiles of this range represent the low-, intermediate-, and high transition readiness levels based on viral suppression one year after transitioning to adult care. We then assessed the performance of the transition readiness tertiles by calculating the sensitivity, specificity, positive and negative predictive values of the high and low transition readiness categories compared to the other two tertiles.We used a point scoring system to develop the final transition readiness score . First, We used a scoring method of converting the regression coefficient for each predictor into an integer. We then multiplied the integer by -1 to easily present transition readiness as predicted probability of viral suppression in practice. To assess model goodness of fit , we constructed a calibration plot of the two outcomes. Perfect calibration is achieved when points on the graph lie on or around the 45-degree line. We also performed the Homer-Lemeshow Goodness of fit test where p > 0.05 indicates good calibration . To eval Biomedical Research Ethics Counsel of the University of KwaZulu-Natal, KwaZulu-Natal Department of Health, Partners Healthcare/Massachusetts General Hospital Research Ethics Board and the Emory University Institutional Review Board approved this protocol.We prospectively enrolled 199 adolescents with perinatally-acquired HIV during their last visit in the pediatric clinic. Among these participants, the median age at transition was 15 years (range 12–21 years), the median age at ART initiation was 8 years (IQR 5–9 years), and 98 (49%) were female as indicated in Table Prior to transition 147 (74%) of adolescents had viral suppression, while one year after transition to adult care, 84 42%) of adolescents had viral suppression. On bivariate analysis, adolescents on first-line ART had higher odds of suppression after transition  compared to those on second-line ART. Conversely, adolescents who had ever used alcohol  or were older at ART initiation  were associated with lower odds of suppression after transition to adult care. On multivariable analysis, being on first-line ART , having documented HIV status disclosure , and higher HARTS  were associated with a higher odds of viral suppression after transition, while females , those with older age at ART initiation , and those who had ever used alcohol  were associated with a lower odds of viral suppression after transition as indicated in Table % of adolUsing the risk factors associated with viral suppression after transition to adult care in the multivariable model including age at ART initiation, ART regimen, HIV disclosure, HARTS, Alcohol use, and biological sex Table , we creaFor the final model, the Hosmer-Lemeshow p value was 0.36 and the calibration plot fell along the 45-degree line suggesting good agreement between the predicted and actual probability of viral suppression as indicated by Fig. The transition readiness score ranges from − 8 to 11. Participants scoring ≥ 5 are considered to have high transition readiness with the highest odds for viral suppression one year after transition to adult care. Those scoring between − 1 and 4 are at intermediate transition readiness, while those scoring ≤-2 have low transition readiness and have the lowest odds for viral suppression after transition. The overall sensitivity of the intermediate/high transition readiness category (>-2) compared to low transition readiness category (≤-2) in determining viral suppression one year after transitioning to adult care was 96.4% (95% CI 89.9–99.3) with a specificity of 27.7% (95% CI 19.6–36.9) and a negative predictive value of 91.2% (95% CI 76.3–98.1) positive predictive value of 50.0% (95%CI 42.1–57.9) as indicated in Table We present the development of a transition readiness score through a prospective analysis of factors associated with viral suppression among adolescents living with perinatally-acquired HIV transitioning to adult care. To our knowledge, this is the first transition readiness score assessment for this population. This clinical prediction tool can be useful to clinicians by providing evidence-based decision support around timing of transition to adult care for adolescents living with perinatally-acquired HIV in South Africa and potentially in other similar settings. By creating high, intermediate, and low transition readiness tertiles, clinicians through the administration of a brief questionnaire evaluating drug use and transition readiness (HARTS) combined with demographic information , can determine which adolescents are likely ready for transition to adult care and which adolescents may need additional services prior to transition.Based on the sensitivity and specificity analysis, we suggest using the transition readiness score in addition to clinical judgement to determine timing of transition from pediatric to adult care. We favor the high specificity of scoring ≥ 5 and its significant association with viral suppression after transition as the binary threshold. Therefore, adolescents scoring in the high range (≥ 5) are likely ready to transition to adult care without additional intervention with a high likelihood of viral suppression one year after transition. Adolescents scoring in the intermediate and low range (< 5) would likely benefit from additional time in pediatric care or targeted interventions to address modifiable areas of deficiency. We plan to use this assessment in future studies to provide differentiated care for adolescents at higher risk of failure during transitioning to adult care.Currently, there are no clear guidelines on when to transition adolescents and how to best determine transition readiness for adolescents living with HIV in South Africa . In mostWe found that the multidomain transition readiness assessment  carried significant weight in predicting viral suppression after transition to adult care . Other tWe found that disclosure, the events leading an adolescent to becoming aware of their own HIV status, was important to transition readiness. In this study, disclosure was evaluated by two separate methods. Disclosure was first evaluated as one of the HARTS domains to evaluate self-assessed knowledge of disclosure and second by documentation in the medical record to evaluate clinical team’s assessment of and involvement in the adolescent’s understanding of their diagnosis. Without knowledge of their HIV status, adolescents cannot participate in peer support activities that can adversely affect self-esteem and mental health resulting in decreased engagement in care. Disclosure has direct effects on internalized stigma, mental health and other factors that can assist adolescents during transition to adult care , 27. DocWithout additional intervention, adolescents who have already failed first-line ART treatment continue to have high rates of failure likely due to ongoing poor adherence or ART resistance , 32. AltThis transition readiness score is designed for adolescents who are not using drugs. Drug use has been associated with poor adherence and viral failure among adolescents in other studies , 36. AltThis study has several limitations. The study was conducted in a single population with a younger age range (median of 15 years old) for transition to adult care. Future studies will be needed to assess transition risk among adolescents who transition at older ages, in settings outside South Africa, and among adolescents with non-perinatally-acquired HIV. We also used convenience sampling to enroll participants, although most eligible participants agreed to participate; this approach could have introduced bias into the sample. However, baseline demographics were similar to other clinics in the region , 20. In We developed a transition readiness score for adolescents living with perinatally-acquired HIV to identify which adolescents are ready to transition to adult care and who may need additional resources prior to transition. Modifiable factors such as transition readiness, disclosure, and drug/alcohol use can be optimized prior to transition to help improve one-year viral suppression outcomes among adolescents after transition to adult care.


Our study aimed to evaluate the stability of human immunodeficiency virus 1 (HIV-1) RNA on cobas plasma separation card (PSC) specimens for viral load (VL) testing after being exposed to varied temperatures and storage times.For this purpose, venous PSC specimens were collected and stored at 25ºC to 42ºC for a period of up to 28 days. Plasma VL at baseline was used as reference, against which PSC VL was compared at different time points.From the 30 patients included in the study, 600 PSC and 30 fresh plasma specimens were obtained. Plasma VL at baseline was fewer than 1,000 copies/mL in 16 patients, and 99.4% of PSCs from these patients yielded nonquantifiable VL at all temperature ranges and time points. During the study period, minor variation of VL was observed in PSCs obtained from 13 patients with plasma VL fewer than 1,000 copies/mL at baseline. For the patient with plasma VL at 1,000 copies/mL, the PSC VL varied from undetectable to 1,670 copies/mL.Our results show minor variation of VL in PSC specimens in the study conditions. HIV RNA is stable in PSCs exposed to high temperatures for up to 28 days. The stability of human immunodeficiency virus 1 (HIV-1) RNA for viral load testing on cobas plasma separation card (PSC) specimens was evaluated after exposure to various temperatures and storage times.HIV-1 RNA is stable in cobas PSC specimens at up to 42°C during 28 days.The cobas PSC specimen is a viable alternative to fresh plasma for HIV viral load determination in hard-to-reach settings.2 and (2) challenges for the fast transportation of specimens from hundreds of primary health care facilities to the testing laboratories.3 Dried blood spots (DBSs) have been widely implemented as a viable alternative specimen for HIV-1 VL measurement.4-8 Advantages of DBSs include the possibility of transportation under easier biosafety conditions at room temperature. However, due to the interference of proviral DNA and intracellular RNA, VL values determined using DBSs are known to be overestimated at the clinically relevant range of 1,000 copies/mL.9-11Monitoring of human immunodeficiency virus 1 (HIV-1) viral load (VL) during antiretroviral treatment is best achieved in fresh plasma. However, the use of this specimen type has been hampered in sub-Saharan Africa due to (1) the scarcity of well-equipped laboratories, which exist only in a few main urban centers,13 Despite this recent evidence of the good performance of the PSC for HIV-1 VL testing, it is unknown whether HIV-1 RNA in the PSC remains stable when exposed to higher temperatures in the tropics. These data are important for the establishment of functional referral systems in sub-Saharan Africa, given the variability of temperature during transportation from clinics to the testing laboratory. Our study aimed at evaluating the stability of HIV-1 RNA for VL testing on the cobas PSC after exposure to various temperatures and storage times.Recently, the cobas plasma separation card (PSC) was shown to be a more accurate alternative to DBSs.HIV-infected adults initiating antiretroviral therapy (ART) in Primeiro de Maio Health Center in Maputo City were consecutively enrolled in the study in July 2019. Beside the DBS specimens routinely collected for HIV-1 VL monitoring, additional venous blood was collected to prepare the cobas PSC specimens and fresh plasma.13The cobas PSC (Roche Molecular Systems) is a novel device that allows whole blood collection and filtration into dried plasma spots. The device comprises a porous membrane, which retains some blood components and allows plasma to filter through and be collected onto an underlying polyester fleece. The latter is impregnated with an RNA stabilizing reagent. After collection and drying, the specimen on the card can be packed and transported to a laboratory for HIV-1 VL testing.2EDTA and sent to the Instituto Nacional de Saúde laboratory within 6 hours after venesection, where 20 cobas PSC specimens were prepared for each patient. The remnant whole blood was immediately centrifuged at 800 to 1,600g for 20 minutes at room temperature to obtain plasma, which was stored at –80ºC until testing.For each patient, 12 mL of venous blood was collected in a BD Vacutainer KFor the PSC card preparation, 140 μL of whole blood was transferred onto each of the three delineated areas of the card and dried overnight at room temperature. After drying, cards were immediately packed in airtight plastic bags containing desiccant gel and humidity indicators and stored during 1, 3, 7, 21, and 28 days under four time range–controlled temperatures of 2°C to 8°C, 25°C, 37°C, and 42°C up to the testing day. Whenever the color of the humidity indicator changed from blue to pink, both the humidity indicator and desiccant gel were replaced with new ones.For both plasma and PSC specimens, VL was tested using the Roche CAP/CTM 96 HIV-1 Quantitative Test v2 (Roche Molecular Diagnostics), according to the manufacturer’s instructions. Interpretation of VL results was performed according to the manufacturer’s instructions, which establish 20 and 738 copies/mL of viral RNA as the low limit of quantification (LoQ) for fresh plasma and cobas PSC specimens, respectively.10 copies/mL. Specimens with nondetectable VL results and with values below the LoQ, for both plasma and PSC specimens, were assigned a value of 1 copy/mL to enable quantitative log10 copies/mL transformation. Specimen types were categorized by plasma VL results and based on the LoQ of the cobas PSC (738 copies/mL). The stability of nonquantifiable results is reported as percent concordance. The absolute VL result is provided where discordance is noted. The fresh plasma VL result (24-hour specimen) was taken as the reference against which all PSC VL results were compared over time and by temperature category. Bland-Altman analysis was used to measure the mean difference and limits of agreement between plasma VL at baseline and cobas PSC VL for different storage temperatures and time points.14 A difference of less than 0.3 log copies/mL was considered acceptable and had no implications for clinical follow-up.15All VL results were transformed to logEthical approval for the study was obtained from Mozambique’s National Health Bioethics Committee (reference number 297/CNBS). Written informed consent was obtained from each patient prior to conducting any study procedure.Thirty HIV-infected adults initiating ART schemes were enrolled for the study in July 2019. The median age of patients was 39 years, and most (56.7%) were female.TABLE 1.Of 30 patients included in the study, 16 had plasma VL fewer than 1,000 copies/mL: 3 had undetectable plasma VL, 6 had VL fewer than 20 copies/mL, and 7 had VL fewer than 738 copies/mL. One patient had a VL at 1,000 copies/mL, and 13 patients had a VL between 3,000 and 800,000 copies/mL. The 600 PSC cards that were generated from the 30 patients recruited for this study were exposed to various storage temperatures and times Almost all of the PSC cards  from the 16 patients with plasma VL fewer than 1,000 copies/mL at the baseline remained with nonquantifiable VL results at all temperature ranges and time points over the 28 days of the study. A minor proportion of these PSC specimens  generated quantifiable VL values of 1,354 copies/mL and 2,954 copies/mL in two cards obtained from two different patients, which were stored at 37ºC during 21 and 28 days, respectively.The quantification of viral RNA for the patient with plasma VL at 1,000 copies/mL yielded results ranging between target not detected and 1,670 copies/mL FIGURE 1. In general, PSC specimens showed a slight reduction in viral nucleic acid quantities after storage for up to 28 days. Specimens stored at 2ºC to 8ºC A total of 260 PSC cards were generated from 13 patients with plasma VL between 3,000 and 800,000 copies/mL TABLE 1All specimens from the 13 patients with VL values more than 3,000 copies/mL had a variance less than 0.3 log copies/mL at all time points and temperatures of the study, except at day 1 at a temperature of 2ºC to 8ºC and 25ºC, in which the variance was 0.31 log copies/mL.15The mean differences displayed for the various temperature and time points of the study are in line with the tolerable difference of less than 0.3 log copies/mL and should not be of clinical significance for the follow-up of patients.The use of plasma for measuring HIV-1 VL in resource-limited settings poses formidable logistical challenges. Although the introduction of DBSs has contributed to increased access to HIV-1 VL testing, this specimen type yields inaccurate results due to the presence of proviral DNA and RBC-associated viral RNA. The novel PSC retains both the logistical advantages of DBS specimens and the accuracy of plasma for HIV-1 RNA measurement.13 Additional data from a similar study recently conducted in Mozambique yielded a sensitivity of 99.8% (99.8%-100%) and 100% (99.2%-100%) in capillary and venous PSC specimens, respectively. The specificity was 97.3% (92.4%-99.4%) in capillary PSC specimens and 98.2% (93.6%-99.8%) in venous PSC specimens. The misclassification rate for this study was 0.7% in capillary PSC specimens and 0.3% in venous PSC specimens.12Viral loads determined on PSC specimens have been shown to be in agreement with those measured in fresh plasma. A study conducted in South Africa to seek the performance of PSCs to identify virologic failure at the threshold of 1,000 copies/mL yielded a sensitivity of 97.0% (92.4%-99.2%) and a specificity of 97.2% (94.9%-98.6%). The misclassification rate in this study was 2.9%.15 Our findings are in line with results from a previous study that showed that the cobas PSC specimen can be stored for 56 days between 2ºC and 30ºC, without significant variance on the HIV-1 VL result.13Our results further show that plasma HIV-1 VLs are in close agreement with those generated from PSC specimens stored for up to 28 days after collection. Moreover, PSC storage at temperatures as high as 42ºC does not negatively affect the accuracy of HIV-1 VL. The discrepant results observed in two cards that showed nonquantifiable VL at baseline but generated quantifiable VL results when stored at 37ºC during 21 and 28 days, respectively, may be due to deficiency on the porous membrane of those specific cards, which compromised the retention of blood components during specimen preparation. This could cause incorrect categorization of viral suppression and potentially lead to an unnecessary switch of the ARV scheme. However, general data from our study showed a minor decrease of the VL values, with no clinical significance across all storage temperatures and time points of the study.Humidity is another factor that can influence stability of biological specimens. However, humidity conditions were not evaluated in the present study. As a standard procedure in sub-Saharan Africa, DBS and PSC cards are stored and transported in airtight plastic bags containing desiccant gel and humidity indicators. This practice has been adopted to mitigate the negative effects of the humidity.12 The stability of PSC specimens stored at high temperatures for up to 28 days is an important feature for field conditions in resource-limited settings, given that in most sub-Saharan African countries, preshipment storage at health facilities and transportation of specimens to the reference laboratories can take up to a month.16It is also important to note that the higher LoQ of the cobas PSC specimen (738 copies/mL) compared with fresh plasma (20 copies/mL) requires more frequent VL monitoring at 3-month intervals, especially for patients with VL lower than 1,000 copies/mL. Nevertheless, the cobas PSC specimen has been shown to be suitable to identify virologic failure and treatment success at this clinically relevant threshold.In addition to the good performance for VL quantification reported in previous studies, this study demonstrated that HIV VL can be accurately determined in PSC cards stored at temperatures as high as 42ºC for up to 28 days. Taken together, these data support the use of the cobas PSC as a good alternative to fresh plasma for the laboratory monitoring of ART in resource-limited settings.


Although the mortality rate among individuals diagnosed during the pre-Highly Active Antiretroviral Treatment era has been substantial, a considerable number of them survived. Our study aimed to evaluate the prevalence of HIV long-term survivors in a cohort of People Living with HIV diagnosed between 1985 and 1994 and to speculate about potential predictive factors associated to long survival. This is a retrospective single-center study. Subjects surviving more than 300 months (25 years) from HIV diagnosis were defined as Long Term Survivors. Overall, 210 subjects were enrolled. More than 75.24% of the included people living with HIV were males, with a median age of 28 years (IQR 25–34). The prevalent risk factors for HIV infection were injection drug use (47.62%), followed by unprotected sex among heterosexual individuals (23.81%). Ninety-three individuals (44.29%) could be defined as LTS with a median (IQR) survival of 333 (312–377) months. A hazard ratio of 12.45 (95% CI 7.91–19.59) was found between individuals who were exposed to Highly Active AntiRetroviral Treatment (HAART) and individuals who were not, with the latter being at greater risk of death. The availability and accessibility of effective antiretroviral therapy for people living with HIV remain the cornerstone of survival.  Since the beginning of the epidemic, the human immunodeficiency virus (HIV) radically changed lives of millions of women and men in the world. The World Health Organization (WHO) estimated that in 2021, 38.4 million people were living with HIV infection, and 650,000 people died because of HIV . NowadayMany factors have been associated with survival in PLWH. Adherence to an effective antiretroviral (ARV) treatment and the degree of immunodeficiency at the time of combined antiretroviral therapy (cART) initiation seem to be the most important ones ,13. CD4+Understanding the factors that contribute to long-term survival is crucial for providing further insight into the natural history and pathophysiology of HIV infection, for comparing the mortality rates in PLWH with those of the general population, and for developing longer-term survival models. The aim of this study is to evaluate the prevalence of LTS in a cohort of PLWH diagnosed between 1985 and 1994 and to speculate about potential predictive factors associated with long survival. Therefore, we evaluated epidemiological and clinical parameters recorded at the time of diagnosis.We conducted a retrospective observational monocentric study. All subjects with a diagnosis of HIV infection confirmed with a Western Blot assay between 1 May 1985 and 31 December 1994 in follow-up at the Infectious Diseases outpatient clinic of the Garibaldi Nesima Hospital in Catania, Italy, were considered eligible for the study. This research was conducted according to the Declaration of Helsinki. Subjects signed a general written informed consent to the use of their data for research purposes upon the admission. The data were collected by a member of the medical team that treated each individual, and these data have been codified and anonymized before their statistical analysis. For the retrospective design of the study, based on a database enquiry and medical records consultation, we did not request ethical committee approval.Epidemiological characteristics: sex, age, HIV exposure .3/µL), creatinine (mg/dL), aspartate aminotransferase (UI/L), alanine aminotransferase (UI/L); date of most recent visit; antiretroviral therapy before the first cART; data of starting cART; AIDS-defining illnesses; AIDS presenter; date and cause of death.Clinical parameters: date of HIV diagnosis; baseline CD4+ T-cells count (cells/µL); CD8+ T-cells count (cells/µL); CD4+/CD8+ ratio; CD4+ nadir; HIV-RNA viral load (copies/mL), if available; HBsAg and HCV antibody test results; the first and the last recorded laboratory tests, including hemoglobin (g/dL), platelets count , using BD Multitest™  and four-color direct immunofluorescence reagent according to the manufacturer’s instructions. The BD Multitest™ includes CD3 fluorescein isothiocyanate (FITC), CD8 phycoerythrin (PE), CD45 peridinin chlorophyll protein (PerCP), CD4 allophycocyanin (APC). CD4® HIV-1  according to the manufacturer’s instructions. The lower limit of detection is 20 cps/mL; the higher limit of detection is 107 cps/mL.Plasma viral load was measured in 400 µL of plasma with CobasThe effectiveness of ART was evaluated by the achievement of an undetectable plasma HIV-RNA viral load (<50 copies/mL) since its measurement became available. Longitudinal observation was stopped on 31 December 2019, that is, before the beginning of the SARS-CoV-2 pandemic. Subjects surviving more than 300 months from HIV diagnosis were defined LTS.All subjects with an ELISA antibody test positive for HIV, as confirmed with a Western Blot assay between 1 May 1985 and 31 December 1994.Age > 18 years.PLWH who were lost to follow-up or transferred to another ID center before completing the entire observation period (31 December 2019).Individuals who received the first diagnosis elsewhere.Continuous variables have been presented as median and interquartile range (IQR), whereas the categorical variables have been expressed with frequencies and percentages. In the evaluation of the parameters that were stated as not normally distributed thanks to the Shapiro–Wilk test of normality, the non-parametric Wilcoxon rank-sum test was applied. The Kaplan–Meier estimator was used for survival analyses building survival curves. Hazard Ratio was estimated through Cox proportional hazards regression model. To understand the relation between variables and survival, we used linear regression. Statistical analysis was performed with jamovi 2.0.0.0 for MacOS and Graphpad Prism 9.4.1 .A total of 224 subjects were initially identified; of these, 14 individuals were excluded because they were lost to follow-up soon after the diagnosis. Overall, 210 PLWH, in follow-up at the Infectious Disease Department at Garibaldi Nesima Hospital in Catania, were enrolled. Characteristics of the cohort are summarized in Overall survival is shown in Median time of survival for this cohort was 209 months (IQR 31–328). At the end of the period of observation, 90 individuals (42.9%) were still alive. The main cause of death was an AIDS-related event . We divip = 0.034). This difference is even more significant when comparing NLTS and LTS: almost none of the LTS individuals was an AIDS case at diagnosis (p < 0.001)  .p < 0.001) between the respective median time of survival. In fact, median time of survival in HAART-treated individuals was 316 months (IQR 300–369), whereas median time of survival in no-HAART subjects was 24 months (IQR 9–46). Overall, 120 PLWH in this cohort were on HAART treatment (57.1%), whereas 90 were not (42.9%). Therefore, we decided to compare overall survival in patients not undergoing HAART treatment vs. patients who took HAART. Hazard ratio, measured with a log-rank method, was 12.45 (95% CI 7.91–19.59), with individuals who were not exposed to HAART being at greater risk of death. We also discovered a statistically significant difference  had access to HAART. We compared the effects of other factors on differences of survival of these sub-cohorts. Moreover, only two PLWH received HAART after AIDS diagnosis, one LTS and one NLTS.p = 0.154) in terms of survival. However, when we stratified PLWH by mode of transmission, it came out that having a CD4+ T-cell count < 200 cells/µL was significantly associated with a shorter survival (p < 0.001). We also stratified both LTS- and NLTS-PLWH by CDC ’93 CD4+ T-cell category, and we did not find any statistically significant difference between survival rate in these sub-groups . Firstly, we chose to stratify the whole subgroup of PLWH having access to HAART by CD4+ T-cell count at diagnosis. There is no statistically significant difference between having a CD4+ T-cell count at diagnosis higher than 500 cell/µL, between 200–500 cells/µL, or lower than 200 cells/µL , although a certain trend towards shorter survival can be seen, especially in NLTS-PLWH with lower CD4+ T-cell counts at diagnosis (p = 0.351), even when stratifying by CDC ’93 category . The difference was not statistically significant, even when stratifying for a CD4+/CD8+ ratio higher (p = 0.159) or lower than 0.5 (p = 0.634) (We then studied the differences in terms of CD4+/CD8+ ratio between LTS- and NLTS-PLWH. We did not find any differences between the median CD4+/CD8+ ratio in LTS and NLTS-PLWH (= 0.634) .p = 0.19) in terms of survival. Taken together, these data show that CD4+/CD8+ ratio at diagnosis is not a good predictor of long-term survival in patients taking HAART.p = 0.59), of having HCV co-infection (p = 0.27), and of having HBV co-infection (p = 0.84). Although having AIDS at diagnosis was a statistically significant predictor of mortality in the whole cohort, this significance was not confirmed when only PLWH having access to HAART were considered (p = 0.79).We also evaluated the effect on survival time of antiretroviral treatment before HAART  (p < 0.001) and those who acquired the infection through unprotected heterosexual sex (p = 0.027).However, age at diagnosis influences time of survival (< 0.001) . These dSince the beginning of the epidemic, around 84.2 million people worldwide have been diagnosed with HIV, and 40.1 million of them are estimated to have died from AIDS-related illnesses . Many stOur analysis showed a robust association between receiving a cART and survival. This is not surprising given that studies suggest that early effective antiretroviral therapy delayed death and transformed HIV infection from being a fatal disease into a chronic condition. Many studies have found that AIDS-defining illnesses as the cause of death are declining dramatically after HAART introduction . Our anaIn our cohort, the main risk factor for HIV infection was intravenous drug use (47.62%). These data are consistent with previous studies . IDUs haMany studies have demonstrated that HIV/HCV coinfection is related to a rapid progression of the liver damage showing the benefit of the HCV eradicative therapy for the health of PLWH; however, we did not find any association between HCV and survival rate . We collIn our study, male sex and younger age are associated with a more favorable prognosis in terms of long-term survival. Nowadays, early diagnosis of the infection is the key to successful management of HIV, allowing for prompt initiation of an antiretroviral regimen and lowering the risk of disease progression . The proThe goal of modern ART is to obtain persistent undetectability of HIV-RNA plasma viral load  and immune reconstitution . Persons initiating ART at very low CD4+ cell counts are at higher risk of opportunistic infections and death . These POur findings support the results obtained by Ren et al. . They shMany studies have established a clear association between lower total CD4+ cell counts and the development of opportunistic infections in HIV subjects ,27. The Our study has some limitations that should be addressed. Firstly, this is an observational, retrospective analysis. Furthermore, it is a single-center study, and it consists of a limited sample size. Finally, some data about clinical and laboratory status of PLWH were missing in the medical records. A possible bias in our analysis is that not all the subjects began treatment at the same time after diagnosis; moreover, they were diagnosed at vastly different stages of infection, and almost none of them ever had any testing before diagnosis. In addition, we do not have any data about adherence to treatment. Many of them were already dead by the time HAART was available. However, it also has some strengths. First of all, the data collection was performed in a single center in which the medical staff have operated since 1985, at the beginning of the HIV pandemic in our area, with an exciting blend of memories, dusty paper archives, and rudimentary electronic files. Moreover, the retention in care of such a high percentage of subjects (93.7%) in the same clinical center for such a long time has been crucial for a complete data collection.This retrospective observational study showed that 44% of PLWH diagnosed during the first decade of the HIV pandemic survived more than 25 years from their first diagnosis. This was an unimaginable result at the beginning of the pandemic, and to share this information could considerably push the screening campaign and the access to care for unaware or hesitant people. Moreover, it confirmed that conditions traditionally associated with late presentation, such as older age and lack of ARV treatment, are associated with bad prognosis. This study also demonstrates declining death rates among HIV-infected persons in our cohort with access to HAART. The availability and accessibility of effective antiretroviral therapy for people living with HIV remain the cornerstone of survival. Prompt initiation of ART and adherence to the treatment remain the stronger predictors of outcome. Therefore, early diagnosis and linkage to care of PLWH play a key role in the HIV care continuum and represent the necessary first steps to antiretroviral therapy initiation and viral suppression, with the purpose of reducing the risk of developing HIV transmission, HIV-related complications, and death.


Integrase inhibitors (INIs) are a potent option for HIV treatment. Limited data exist on INI resistance in West Africa, particularly in children living with HIV/AIDS. We determined the prevalence of integrase gene polymorphisms and the frequency of naturally occurring amino acid (aa) substitutions at positions associated with INI resistance. Dried blood spot (DBS) samples were obtained from one hundred and seven (107) HIV-1-infected children aged less than 15 years old in two West African countries, Benin and Mali. All children were naïve to INI treatment, 56 were naïve to anti-retroviral therapy (ART), and 51 had received ART. Genetic sequencing of HIV integrase was successful in 75 samples. The aa changes at integrase positions associated with INI resistance were examined according to the Stanford HIV Genotypic Resistance database. The median ages were 2.6 and 10 years for ART-naïve and -treated children, respectively. The most common subtypes observed were CRF02_AG (74.7%) followed by CRF06_cpx (20%). No major INI-resistance mutations at positions 66, 92, 121, 143, 147, 148, 155, and 263 were detected. The most prevalent INI accessory resistance mutations were: L74I/M  followed by E157Q , G163E/N/T/Q , Q95A/H/P , and T97A . Other substitutions observed were M50I/L/P, H51E/P/S/Q, I72V, T112V, V201I, and T206S. Polymorphisms at positions which may influence the genetic barrier and/or drive the selection of specific INI-resistance pathways were detected. However, no transmitted drug resistance (TDR) to INI was detected among samples of INI-naïve patients. These findings support the use of this treatment class for children with HIV-1, particularly in West Africa. Major efforts have been directed towards the development of drugs targeting HIV-1 integrase, a crucial enzyme involved in HIV-1 replication . First-gAn estimated 1.8 million children aged 0–14 were living with HIV at the end of 2019, and 150.000 children were newly infected. The sub-Saharan African region remains the most affected. Benin and Mali, two West African countries, have experienced similar HIV-1 epidemics, with prevalence rates of around 1% ; the most prominent strain circulating within these countries is CRF02_AG [A multi-country analysis of HIV drug resistance in sub-Saharan African countries showed that 53.0% and 8.8% of newly diagnosed infants had resistance to one or more non-nucleoside reverse-transcriptase inhibitors (NNRTIs) and nucleoside reverse-transcriptase inhibitors (NRTIs), respectively, before treatment initiation . The WorThe ODYSSEY trial, a randomized study that included African countries, showed evidence of the superior efficacy of dolutegravir-based ART, compared with standard care in children and adolescents, as first-line and second-line ART .http://hivdb.stanford.edu, accessed on 2 January 2023) based on their associations with reduced drug susceptibility: (1) major mutations represent non-polymorphic mutations that, by themselves, reduce susceptibility to one or more INIs; (2) accessory mutations represent polymorphic or non-polymorphic mutations that can reduce INI susceptibility in combination with major RAMs; (3) other mutations are polymorphic or non-polymorphic mutations that can be selected under INI-based therapy and which may or may not potentially reduce susceptibility [Since the implementation of INI regimens, surveillance of INI-selected mutations has gained in importance . Severalibility:  major muIn developed counties, INI RAMs have been well studied but there are few data for Africa, particularly in children. The aim of this study was to characterize the prevalence of HIV-1 natural polymorphisms and RAMs in INI-naïve, HIV-1-infected children in two West African countries.Dried blood spots (DBSs) were obtained from 107 HIV-1-infected children aged less than 15 years old living in two West African countries, Mali and Benin. The DBSs were collected from 51 ART-treated children with virological failure, defined as having at least one viral load (VL) >1000 copies/mL, during a study conducted in Benin between 2015 and 2016  and fromhttp://www.hivfrenchresistance.org/, accessed on 2 January 2023). HIV-1 RNA was extracted from the 107 DBSs using EasyMag technology as per the manufacturer’s instructions. Target integrase regions of the HIV-1 were subsequently amplified by PCR after reverse-transcription production of complementary DNA (cDNA) and sequenced using the Sanger method. Genotypic-resistance testing was conducted on the integrase gene’s target regions of HIV-1 using the ANRS ((French) National Agency for Research on AIDS and Viral Hepatitis) method : 45′ at 45 °C, 2′ at 95 °C, then:  × 40 cycles. Nested PCR step, using Ampli Taq Polymerase with Gene Amp (Roche): 5′ at 94 °C, then ; 1′ at 72 °C) × 45 cycles 7′ at 72 °C.https://hivdb.stanford.edu/hivdb, accessed on 2 January 2023), with a single integrase gene amplification. Regarding INI mutations, all mutations reported in the IAS and Stanford lists were considered; in particular, the major INI RAMs from the latest version of Stanford’s list, including T66A/I/K, E92Q, G118R, E138K/A/T, G140S/A/C/R, Y143R/C/H, S147G, Q148H/R/K, N155H, and R263K . Some uncommon nonpolymorphic accessory mutations, such as M50I/L/P, H51Y, and F121Y, alone, do not reduce INI susceptibility [Seventy-five (75) of the 107 samples were successfully sequenced, assembled, and aligned using Sescap and SmartGene HIV Software . The other 32 samples could not be amplified. The INI sequences, successfully analyzed from 75 DBS samples, were from 31 ART-treated children with virological failure  and 44 ART-naïve HIV-1-infected Malian children. Positions associated with INI resistance were analyzed, and the interpretation of INI resistance was performed following the Stanford HIV resistance algorithm, version 7.0. The HIV-1 subtypes and CRFs were determined according to the Stanford University HIV Drug Resistance Database HIVdb Program, latest version . All statistical tests were two-tailed, with α = 0.05.The characteristics of the patients providing the 75 samples that were successfully sequenced are shown in No residue substitutions of major INI RAMs at codons 66, 92, 121, 143, 147, 148, 155, or 263 were observed in the 75 sequences. Of the 75 sequences, 25/75, 33%, harbored at least one INI accessory polymorphism mutation  and 48 (64%) harbored at least one polymorphism mutation, described as having no impact on INI sensitivity [Among the detected INI accessory mutations, L74I/M was the most prevalent , followed by E157Q , G163EN/T/Q , Q95A/H/P , and T97A . The lowest prevalence of residue substitutions was found at codons G118V, S153F, and S230N, all with the same prevalence (1.3%), representing only 1 of the 75 samples. The prevalence of E138Q was 2.6% (2/75) . Some naThis first report from Benin and Mali found that no major INI RAMs were identified, consistent with prior studies ,14,18, fThe mutation T97A is a naturally occurring and lowly prevalent integrase polymorphism that emerges more frequently among patients carrying non-B, as in our setting, compared to those carrying HIV subtype B ,33,35,36Some substitutions with no known effect on INI susceptibility were also identified, , with prevalences from 5% to 75%. Such frequencies can be attributed to natural genetic variability, frequently observed in the integrase gene ,44. The p = 0.0035) prevalences observed for other polymorphism mutations in naïve and treated patients, respectively. The presence of these mutations in ART-treated though INI-naïve patients is conceivable, since reports have shown that integrase mutations increase their prevalence in ART-treated compared to ART-naïve patients, suggesting that some NNRTIs, NRTIs, or PIs could interact (directly or indirectly) with the integrase inducing or selecting INI secondary mutations [We have reported a higher prevalence of some accessory INI-resistance mutations in ART-treated samples compared to untreated samples. Indeed, 15% of naïve patients and 11% of treated patients harbored at least one INI accessory polymorphism mutation at described positions. These prevalences were statically significant, with 38% and 17% (utations . Howeverutations . Althougutations , they arutations . We alsoutations .Currently, INI-based treatments are associated with excellent clinical results in patients, leading to a healthier lifestyle and reducing the risk of viral transmission. No TDR to INI was detected among the sequences from INI-naïve child patients. These data support using this class of ARTs in West Africa, including in the pediatric population. However, continued surveillance of INI RAMs is recommended to prevent the emergence of resistance to this class of drugs.


Since the onset of the HIV-1/AIDS epidemic in 1981, 75 million people have been infected with the virus, and the disease remains a public health crisis worldwide. Circular RNAs (circRNAs) are derived from excised exons and introns during backsplicing, a form of alternative splicing. The relevance of unconventional, non-capped, and non-poly(A) transcripts to transcriptomics studies remains to be routinely investigated. Knowledge gaps to be filled are the interface between host-encoded circRNAs and viral replication in chronically progressed patients and upon treatment with antiviral drugs. We implemented a bioinformatic pipeline and repurpose publicly archived RNA sequence reads from the blood of 19 HIV-1-positive patients that previously compared transcriptomes during viremia and viremia suppression by antiretroviral therapy (ART). The in silico analysis identified viremic patients’ circRNA that became undetectable after ART. The circRNAs originated from a subset of host genes enriched in the HDAC biological pathway. These circRNAs and parental mRNAs held in common a small collection of miRNA response elements (MREs), some of which were present in HIV-1 mRNAs. The function of the MRE-containing target mRNA enriched the RNA polymerase II GO pathway. To visualize the interplay between individual circRNA–miRNA–target mRNA, important for HIV-1 and potentially other diseases, an Interactive Circos tool was developed to efficiently parse the intricately competing endogenous network of circRNA–miRNA–mRNA interactions originating from seven circRNA singled out in viremic versus non-viremic patients. The combined downregulation of the identified circRNAs warrants investigation as a novel antiviral targeting strategy. Circular RNAs (circRNAs) are generated during backsplicing, a form of alternative splicing . Genes eBacksplicing is a co-transcriptional process that is competitive with canonical splicing . Recent Antagonism of RNA–mRNA networks is a hallmark of viral pathogens infecting hosts within the plant and animal kingdoms and has been attributed to viral RNA silencing suppressor (RSS) activity . An examPhysiological roles for circRNAs have been assigned to infection, most recently to SARS-CoV2 ,15, and Zhang et al. identified circRNAs in the PBMC of three people in the early stages of HIV-1 infection (<180 days since diagnosis) naive to antiretroviral therapy (ART) and three healthy controls by Illumina sequencing of cDNA libraries generated without selection of poly(A) RNAs . The stuIn El-Diwany et al. (2017) , whole bWe downloaded the raw data and performed bioinformatics analysis using the resources of Minnesota Supercomputing Institute . FastQC was run to generate sequence quality plots for each FASTQ file . The Illp < 0.05. The database CircAtlas 2.0 annotated circRNAs by gene locus of origin and predicted complementarity with host miRNAs [http://mirdb.org/, last accessed on 8 August 2021) [https://www.genecards.org/, last accessed on 8 June 2021). Cytoscape software (3.9.0) was used to visualize miRNAs–mRNA interaction networks [Panther was used to run the Gene Ontology (GO) enrichment analysis  with a st miRNAs , which ut miRNAs . The Micst 2021)  identifinetworks . shinyCinetworks . The codThe sensitivity of circRNA detection is dependent on sequencing depth with commercial sequencing providers suggesting >40 million reads per sample. Published RNAseq research has shown that circRNAs can be mined already from 20 million paired-end reads ≥100 bp  for all reads was 36. Since Q-score ≤ 30 predicts 1 in 1000 base calls is incorrect, any bases below Q30 were discarded in the quality control process. The Q-scores: forward reads, 37, and backward reads, 35, corresponded to a >99.9% accuracy in inferred base calling. Therefore, the Q-score parameter was suitably robust to proceed with the analysis.Deduplication is a default step in processing raw reads and preparing the data set for downstream analysis. In the patient data set, 77% of all reads qualified as distinct reads, in which no two sequences were identical. The value serves as a measure of library diversity. If a library is diverse, most sequences will occur only once. Duplicates may arise as PCR artefacts during the template amplification step prior to actual sequencing, optical artefacts detected by the machine or can be biological duplicates. The average guanosine cytosine (GC) content for all reads in the data set was 45%. Regions having 50–60% GC content received the highest coverage, while regions with 70–80% or 30–40% GC content exhibited decreased coverage . Thus, Gr = ≥0.03 [r ≥ 0.03 and identified 1014 reads and 876 reads in the pre-ART and post-ART patients, respectively. The Tier 3 criteria enriched high abundance candidate circRNA and significantly reduced the frequency of circRNA. We concluded the collection of circRNA loci identified by the previously established Tier 2 stringency criterion [The CIRI benchmark criteria identified a total of 7053 and 7411 circRNA reads in the pre-ART and post-ART groups, respectively. Next, we applied additional stringency criteria to stratify the data. Tier 1 was the CIRI2 default criteria, PCC > 2, BSJ > 1, which requires at least one BSJ composed of nonlinear 3’ and 5’ splice sites and predicts circRNA are less abundant than linear reads. Tier 2 stringency criteria were developed by Gao et al. and specified BSJ ≥ 3,  = ≥0.03 . Tier 2 riterion  were appTier 2 circRNAs identified in the pre-ART samples were almost evenly located on the sense and antisense DNA strands A. The vaThe circRNAs observed in the pre-ART treatment were most commonly derived from chromosomes 1 (9.5%) and 2 (8.7%), which are the two largest chromosomes in the human genome . The most prominent category was “histone deacetylation”, having 18-fold enrichment in the pre-ART group . The circRNA upregulation was observed for ARL8B, ATG2B, ATG7, ATG14, CALCOCO2, LAMP2, MFSD8, STX17, TBC1D5, TECPR1, UFL1, and VPS4A. Autophagy is an intracellular degradative process used for recycling proteins from the cytoplasm to generate macromolecular building blocks and energy under stress conditions, to remove damaged organelles, and to maintain cellular homeostasis by playing a role in both innate and adaptive response to viral infection [Upon investigating the parental genes in the post-ART patients, the macroautophagy process was the GO term showing the most significant fold enrichment  and the corticosteroid receptor signaling pathway . Histone methylation is associated with active provirus transcription, implicating the upregulation of these circRNA in the epigenetic regulation of host or viral gene transcription [The most significant GO terms that were common to pre- and post-ART patients were histone H3-K36 methylation . While finding a single significantly upregulated circRNA was unexpected, this observation corresponds to the trends in mRNAs from the same data set. El-Diwany et al. observed that not a single mRNA was differentially expressed pre- vs. post-ART across the 19 individuals in the cohort using a standardized approach for the analysis [After categorizing the circRNA genes of origin, we applied a bifurcated approach to identify circRNA candidates of interest. First, we employed a negative binomial distribution model  and idenanalysis . The resThe circSATB1-as is an 897 bp exonic sense-strand circRNA located on human chromosome 3 that originates from the gene encoding SATB1 long non-coding RNA. The antisense strand of the parental gene encodes SATB1 protein  that plays a key role in T-cell development, and it is responsible for the maintenance of chromatin architecture, providing a specific binding site for chromatin-remodeling enzymes such as HDACs. Interaction between Tat and SATB1 has been investigated in HIV-1-infected T cells and indicate that Tat competitively displaces SATB1-bound HDAC1 thus increasing the acetylation of the promoter . SATB1 wThe second approach to evaluating the data set identified the prevalence of circRNAs in the patient population pre- or post-ART treatment. The unique circRNAs identified in the patient cohort are summarized in Seven circRNA were common to all viremic patients and these seven were not detectable in all post-ART-patients . Those cThe upregulation of parental gene ATXN1 has been shown in CD8+ cells of HIV-1 positive patients . ANKRD17The seven circRNA varied in mature length from 620  to 1634 bp  with the length of circATXN1 being undocumented. With the exception of circHIPK3, which is reported to be highly expressed in skeletal muscle, these circRNAs are abundantly expressed in the spleen, a major reservoir of CD4+ T-cellsand latently infected HIV-1 during ART . Genome One of the most widely investigated activities of circRNAs is serving as an MRE that sequester miRNAs to regulate post-transcriptional gene expression ,5,6,7. Wp = 5.74 × 10−7)  C. The geOf the MREs in common between circRNA and host mRNA, only six were also in common with HIV-1 RNA . Using tn = 10) observed exclusively in host mRNAs identified a network of 70 genes (p = 5.74 × 10−7), and the remaining top four were the same biological processes observed in We then stratified the host genes containing the six MREs in common with HIV-1 and identified 51 host genes B. There 70 genes . The mosin-silico analysis, we visualized the overlapping interactions between the host gene loci of the circRNA, the MRE of the circRNA in common with host mRNA and HIV-1 mRNAs, and the collaborative interaction between these host components . Upon integration into the host genome, RNAPII is required for transcription of the provirus. HIV-1 has been shown to manipulate the activity of RNAPII intricately through the regulatory protein Tat bound to the trans-activation response (TAR) element [Despite significant progress in the last several decades in the development of ART, pre-exposure prophylaxis for at-risk groups, and preventative vaccine prototypes, the HIV-1–host interface remains inadequately defined. The circRNA identified exclusive to all 19 patients pre-ART generated circRNA–miRNA–mRNA networks that have the potential to promote viral replication. The GO category that was enriched by the circRNA loci in all viremic patients was RNAPII (fold enrichment > 4,  element . Tat bou element . The imp element ,70,71.+ T cells compared to activated CD4+ T cells productively infected with HIV-1 [We generated an accessible network analysis tool Interactive Circos to efficiently trace directed interactions for each circRNA and pairs of miRNA-cognate target mRNA. The constructed competing circRNA–miRNA–mRNA network predicts circRNA sequester miRNAs whose activity diminishes HIV-1 replication . Both mith HIV-1 . The difWith resting T cells being the main reservoir of latent infection, the potential downregulation of circRNA squelching these miRNAs conceivably contributes to latency in these cell populations. After inhibiting these miRNAs with antisense inhibitors in resting CD4+ T cells, authors reported >10 times more HIV-1 particles were generated than from cells treated with negative control inhibitor, verifying miRNA inhibition activates latent virus . Our finAdditional cognate miRNA predicted in this study  were detThe limitation of this in silico analysis is that experiments are warranted to validate the predictions. The top priority is quantifying the levels of the seven top candidates in the patients’ primary T cells, followed by verifying the corresponding candidate proteins’ increase in response to the downregulation of the candidate circRNA. Second, transcriptomics data were not available on the patient samples prior to HIV-1 and HCV co-infection, and HCV replication in the patients’ hepatocytes was not documented either. Predicted in the networks was the ONECUT2 transcription factor that is known to be active in hepatic cells. ONECUT was predicted in the network encompassing circANKRD17–miRNA141-5p, circPHC3–miRNA141-5p, and circSATB1–miRNA-22-5p, but involvement of mir122 was not detected. In closing, host circRNA profiles in viremic patients changed significantly after suppression by ART. Future investigation are warranted to define whether circRNA biomarkers of HIV-1 viremia will be viable therapeutic targets to prevent AIDS. The competing endogenous network pipeline developed in this study and displayed by an Interactive circos plot, visualizes the interplay between individual circRNA–miRNA–target mRNA important for HIV-1 and other potentially other diseases.


Patients who are lost to follow-up while on treatment compromise their own health and the long-term success of antiretroviral therapy (ART) programs. Besides, loss to follow-up (LTFU) increases HIV-related morbidity and mortality. Therefore, this study aimed to determine the incidence of LTFU and its predictors among adult HIV positive patients on anti-retroviral therapy at North Shoa zone public hospitals, Northeast Ethiopia.A retrospective follow up study of 517 people living with HIV/AIDS and attending an ART clinic between 2015 and 2020 was conducted at North Shewa zone, public hospitals. Kaplan-Meier failure function together with log rank test was used to compare failure function. Multivariable Cox proportion hazards regression model was used to determine predictors of LTFU.The incidence density rate of lost to follow up among HIV positive adult on ART was found to be 8.9 per 100 adult years observation . In multivariable cox proportional regression analysis, WHO clinical stage-IV , comorbidity disease , body mass index less than 18kg/m2 , cotrimoxazole preventive therapy , and a low CD4 count  were found to be a significant predictors of lost to follow up.3, had comorbidity disease, and cotrimoxazole therapy were a significant predictors of lost to ART follow up. Therefore, appropriate mitigation measures in the at-risk group need to be instigated to advance retention rate.The current study showed that the incidence rate of loss to ART follow-up was high. Body mass index score less than 18kg/m2, advanced WHO clinical stage, CD4<200cell/mm Human Immunodeficiency Virus (HIV) has been a global challenge for the past three decades. In 2017, nearly 36.9 million People living with HIV (PLHIV) worldwide, of these 35.1 million were adults, and about 19.6 million are in Eastern and southern Africa making up 53.1% of PLHIV in the regionEvidence showed that ART significantly reduced mortality and improved life expectancy of HIV-infected patients, but the attainment depends on regular patient follow-upThe high levels of adherence to ART and retention in HIV care programs is critical to achieve optimal health outcomes among PLHIVSeveral studies showed that the predictors of lost to follow up were daily laborerThe study was conducted in North Shewa Zone, public hospitals, Northeast Ethiopia. North Shewa Zone is one of the 13 Zones in Amhara administrative regional state and the City is Debre Berhan Town located, 695 km from Bahir Dar, capital of the region and 130 km from Addis Ababa, capital of Ethiopia. It is border on the south and the west by the Oromia region, on the North by South Wollo, on the North East by the Oromia zone, and the East by the Afar region. It covers an area of 15,936.13 square kilometersA five-year institution based, retrospective follow up study was conducted at North Shewa zone public hospitals from February 1 to March 15, 2020. All adult HIV/AIDS patients who were newly initiated ART and enrolled at selected north shoa zone public hospitals, ART clinic during the required time (i.e. January 2015 to January 2020) was considered in the current study. Besides, clients who had diagnosis and initiated ART at other health institution and subsequently referred to the respective selected hospital for further treatment was included. On the other hands, HIV positive adult patients, whose medical charts were not found and having incomplete data were excluded from the study.The sample size was determined using double population proportion formula by considering WHO clinical stage as the major predictor variablesA data abstraction format was develop from the standardized ART entry and follow up form currently used by the ART clinics. The data were extracted by reviewing patients ART medical charts. Lost to follow up was confirmed by reviewing medical registration in each hospital. The data abstraction format was examined by senior experts to the area of study for content validity. Data abstraction was designed based on study objectives. This included socio-demographic characteristics, baseline clinical information, and treatment outcomes were obtained from patients' medical charts. These clinical and treatment related predictors were taken in to consideration at starting of ART was used as a baseline value. Data were retrieved from patients' ART chart by trained nurses working in the ART clinic using uniform data abstraction format prepared for the study.st, 2020 at selected four hospitals was retrieved from ART registries. The record of all study participants was selected according pre-specified eligibility criteria. Time to lost to follow up was calculated as the time between the date of ART initiation and the date of lost to follow up. In the current study, HIV positive patients were considered as censored, if transfer to other health care institution (based on reported date or last visit date) or if they were alive at the end of the study period . Before collecting the data, baseline and follow up records were reviewed and the registration was identified by their medical record number. The data collectors who are working at the ART clinic were extracted and reviewed the charts.All available information on patient records were checked then appropriate data extraction format was adopted in English in order to extract all the relevant variables to meet the study objectives. The starting point for retrospective follow-up was the time from first initiation of ART among HIV positive adult patients and the endpoint was date of lost to follow up, death, transfer, date of last contact until January 1st, 2020 which ever come first. The chart of HIV positive adult patients enrolled on ART from January 2015 to January 1To ensure the quality of the data pretest was conducted with 5% of the data abstraction format, to check the hospital documentation system with the agreement of the data abstraction format to the objective of the study. Any error found during the process of checking was correct and modification was made into the final version of the data abstraction format. Training on record review was given to data collectors and supervisors for 01 days before actual data collection task. Besides, the data quality was controlled by designing the proper data collection materials, and through continues supervision. The collected data form was examined for completeness and consistency during data entry, storage, cleaning and analysis. The principal investigator of the study was control the overall activity.Data were coded, cleaned, and entered using EPI-data 3.1 and exported to STATA 14 statistical software for analysis. Kaplan-Meier failure function together with log rank test was used to test for the presence of difference in lost to follow up among categories of covariates. Appropriate descriptive statistics were used based on the nature of the data. Bivariable cox regression analysis was first fitted and those independent variables, with a p-value ≤ 0.25 level of significance, were included in the multivariable analysis. Multivariable cox proportional-hazard regression was fitted at 5% level of significance to determine the net effect of each independent variable on lost to follow up after initiation of ART among HIV positive adult patients. To verify the proportionality assumption in Cox's model, we tested the correlation between survival time and Shoenfeld's standardized residuals. The result of this study was expressed as hazard ratios (HRs) with 95% confidence interval. Finally, the result of the study was presented with text, graph and table.The median age of the respondents was 34 (IQR: 28–40 years) and nearly than half (41 %) of them were under the age category of 25–34 years. With respect to sex of the participants about 265(52.27%) were male. Majority, 347(68.44%) of the participants were urban by residence. Nearly half, 240(47.34%) of the participants were married. Majority, 392(77.32%) of the respondents are orthodox religion follower. One hundred seventy two (33.93%) of the respondents have no formal education and only 20.2% have higher educational level. Regarding occupational status nearly one third, 144(28.4%) has self-employee work. Most of them, 410 (80.87%) have contact person during their follow up care .2. The majority of the participants, 379 (74.75%), were at working functional status. Regarding the WHO clinical stage over one third 194(38.26%) were clinical stage I, while one third, 166 (32.74%) were clinical stage III. Majority, 411(81.07%) of the study participants were disclosed their status to anyone , had opportunistic infections, of these 86 (54.43%) had Tuberculosis, 32 (20.25%) bacterial pneumonia, and 16 (10.13%) other opportunistic infections. Nearly one quarter, 122(24.06%) had comorbidity disease at baseline. Regarding body mass index more than one quarter, 138(27.22%) had less than 18.5 kg/mo anyone .Nearly one third, 156 (30.77%) of the patients were eligible for ART by WHO clinical stage and total lymphocyte count, while 151 (29.78%) were so by CD4 cell count criteria. Cotrimoxazole Preventive Therapy (CPT) was given to 338 (66.67%) study participants. More than half, 278 (54.83%), of the patients started ART treatment with TDF–3TC-EFV and 36 (6%) changed to another regimen within the first line, and while 111(21.89) were used AZT-3TC-NVP regimens. One fourth, 132 (26.04%) of study participants had reported drug side effect .There were 118 cases of LTFU observed over 1,322 person years at risk. The incidence rate of lost to follow up was 8.9 per 100 person years observation . The incidence proportion of lost to follow up in the cohort was 23.27%. The study participants were followed for a median of 32.46 months (IQR: 15.63–45.05) after initiation of ART. The probability of lost follow up among ART patients was 6.32%, 13.16%, 20.74%, 34.24%, and 43.59% at 1, 2, 3, 4, and 5 years of follow up respectively . The oveThis figure shows if the cox regression model fits the data, these residuals should have a standard censored exponential distribution with hazard ratio. If we comparing the jagged line with the reference line, we observe that, the cox model does fit these data to reasonable. The hazard function follows the 45° line very closely. Hence, the output shows cox-snell residuals were satisfied the overall model fitness test .Adults HIV/AIDS patients on ART who were on WHO clinical stage-IV increased the hazard ratio of LTFU by 1.5  times as compared to adults in clinical stages I. Having comorbidity disease at baseline were lowered the hazard ratio for LTFU by 46%  as compared to their counter parts. Body mass index less than 18k/m2 were increased the hazard ratio for LTFU by 60%  as compared with normal weight patients. Those who taken cotrimoxazole preventive therapy were increased the hazard ratio for LTFU by 57%  as compared with don't taken cotrimoxazole therapy. Adults patients who started ART with a baseline CD4 cell count of under 200 cell/m3 were 66% more likely to be LTFU compared to those with a baseline CD4 cell count of greater than 500 cell/m3 , .This study mainly assessed the incidence and predictors of LTFU among adult HIV patients on ART in North Shewa zone, public hospitals, northeast Ethiopia. In this study the incidence rate of LTFU was found to be 8.9 per 100 person year. The finding was consistent with studies done in different regions of EthiopiaOn the other hands, the present study was higher than different studies done in Uganda [20], Debre Markos-Ethiopia2 were  found to be at higher risk of LTFU from adult ART treatment programs using 18.5–24.9 kg/m2 as reference. This finding is consistent with studies conducted in TanzaniaAccording to the present study having a body mass index (BMI) score of less than 18.5 kg/mIn the current study, the hazard of loss to follow among WHO clinical stage IV was 1.5 times higher as compared to clinical stage I . A similar finding was reported by a study conducted in Tanzania3 were more likely to be LTFU. This finding was supported by studies conducted in Yunnan province We observed that patients with baseline CD4 cell count less than 200 cells/mmThe current study found that the risk of LTFU among those who had comorbidity disease was reduced by 46%  compared with those had no comorbidity disease. It is in accordance with the study conducted in NigeriaThe present study had certain limitations that should be taken into account while interpreting these findings. First, we used secondary chart review; excluding of incomplete data may leads to selection bias. Second, patients, who were considered as LTFU might have been died or self-referred to other facilities. Third, since this study used baseline socio-demographic, clinical and treatment related factors, there may be a change of these variables after a time.2, advanced WHO clinical stage, CD4<200cell/mm3, had comorbidity disease, and using of cotrimoxazole therapy were statistical associated to LTFU. Therefore, policymaker and other concerned bodies should implement situation based and context specific preventive measure on these identified factors.In the current study the incidence rate of LTFU among adult HIV/AIDS patients on ART was found to be high. Body mass index score less than 18kg/m