We report a case of dolutegravir resistance in KwaZulu-Natal in a 13-year-old male two years after starting dolutegravir. Resistance most likely developed due to poor adherence as a result of psychosocial issues. This case highlights the importance of the role of the family unit in impacting adherence and close monitoring of treatment-experienced patients with virologic failure following switching to dolutegravir-based regimens. Numerous challenges affect HIV-infected children and adolescents, impacting adherence, as highlighted in this case. The benefit of HIV drug resistance genotyping to guide the selection of a suitable regimen was shown – there is a need for evidence-based guidelines of indications for resistance testing on dolutegravir.1 Various studies have demonstrated that DTG-based ART is highly effective at achieving viral suppression in both ART-naïve and -experienced individuals. Dolutegravir has better tolerability, higher genetic barrier to resistance and is available in a fixed-dose combination of tenofovir (TDF), lamivudine (3TC), and DTG for adults and adolescents ≥ 35 kg.1 In South Africa, DTG is currently available as a 50 mg tablet for children ≥ 20 kg.1 We present the first paediatric case of DTG resistance identified in a treatment-experienced integrase strand inhibitor (InSTI)-naïve adolescent in KwaZulu-Natal.South Africa transitioned to dolutegravir (DTG)-based antiretroviral therapy (ART) from 2019.1 but it is unclear if he received ritonavir super-boosting during his tuberculosis treatment to overcome the inducing effect of rifampicin. He was diagnosed with tuberculosis again in 2013, again without documentation that LPV/r was super-boosted with ritonavir during treatment. Since ART initiation, he has never been virally suppressed, despite ongoing enhanced adherence support.Our index case is a 13-year-old male from a rural area in who acquired HIV through vertical transmission. The mother tested negative during antenatal care, but she was not tested at delivery. He was diagnosed with HIV and pulmonary tuberculosis in 2010, started tuberculosis treatment and later on ART. As per the national guidelines, abacavir, 3TC and ritonavir-boosted lopinavir (LPV/r) syrups were initiated1 HIV VL is in log10 copies/mL  and CD4 count is in cells/µL . HIV VL ≥ 1000 copies/mL (≥ 3 log10 copies/mL) is defined as virological failure.1In 2018 HIV drug resistance (DR) genotyping was performed, showing only M184V mutation with no protease resistance mutations, which suggested suboptimal adherence. He was switched to zidovudine (AZT) and 3TC, and was started on LPV/r paediatric tablets. The patient received continuous enhanced adherence counselling by the attending doctor and via the social worker. In January 2020, LPV/r was switched to DTG in an attempt to simplify the regimen and decrease the pill burden; however, he remained on AZT/3TC as he weighed 28 kg. The VL at the time of the switch was 529 copies/mL and hence a HIV DR test was not performed. However, he remained unsuppressed and admitted to often forgetting his medication as he lacked a treatment supporter.There were major social issues impacting adherence; he is the product of rape by his mother’s older cousin. Being a teen mom, and not having disclosed the rape issue to her mother proved to have affected the patient’s mother, as well as her ability to provide optimum care for her child. She was the primary caregiver living with the maternal grandmother and was responsible for bringing him for his hospital appointments, but would sometimes not be available to bring him, and occasionally the patient’s biological father would accompany the patient to hospital but identified himself as the patient’s uncle. In 2017, the father became ill, and the grandmother (not knowing the issues around the rape) took him into their home, resulting in family conflict and missed hospital appointments. In 2019, both the patient’s biological parents died due to AIDS-related complications. Upon discovering for the first time at her daughter’s funeral that her nephew had raped her daughter and infected her with HIV, the patient’s grandmother struggled to accept the situation. As a result, the grandmother avoided involving herself in the healthcare of her grandson including supervising him when taking treatment, or accompanying him to hospital, making addressing the adherence issues and disclosure difficult, requiring a home visit by the social workers.Due to poor adherence and patient not suppressing for two years on the DTG-based regimen, despite enhanced adherence counselling, HIV DR was done in April 2022 which showed M184V again and the development of InSTI mutations , conferring high-level resistance to DTG refer to .10 drop.The third-line regimen commenced in May 2022 was TDF, emtricitabine and ritonavir-boosted darunavir once daily. The viral load done after one month was 473 copies/mL with a 2 logThis case report highlights the complexities and psycho-social factors impacting the management of HIV in children and adolescents. The paediatric population has unique challenges such as disclosure, caregiver issues, challenges with understanding and acceptance of the disease, limited drug options and child-friendly formulations.In this case, family issues with a lack of supervision resulted in suboptimal adherence, which resulted in the emergence of DTG resistance. Due to his weight of 28 kg in 2020, he was not eligible for a simple once-daily fixed-dose combination of TDF, 3TC and DTG, which may have improved adherence and potentially prevented DTG resistance.2 None of the participants on first-line DTG-based ART with treatment failure had DTG resistance mutations, while 4/22 (18%) participants with virologic failure on second-line DTG-based ART had DTG resistance.2 Similarly, in the IMPAACT P1093 study that assessed DTG in treatment-experienced children and adolescents, 8/36 (22%) participants with virologic failure had resistance to DTG.3Most clinical trials have evaluated DTG in adults. The ODYSSEY (Once-daily DTG based ART in Young people vs. Standard therapy) trial done in children and adolescents found that DTG was superior to standard care with lower rates of treatment failure.4 R263K reduces DTG susceptibility two-fold, conferring intermediate resistance, while G118R reduces DTG susceptibility by > 5-fold, also conferring intermediate resistance.4 Other resistance mutations selected by DTG in InSTI-naïve patients include E138K/T, N155H, Q148K, S230R, T66I and H51Y.4 Accumulation of mutations further reduces DTG susceptibility. These reported mutations are similar to those found in our case.The most common mutations selected by DTG in InSTI-naïve patients are reported to be R263K and G118R.5In sub-Saharan Africa, real-world cohorts have shown high viral suppression rates in countries that have transitioned to DTG. Although DTG is hailed as the silver bullet, it must still be recognised that treatment failure and resistance, especially in treatment-experienced patients with suboptimal adherence, can occur. The potential factors contributing to DTG resistance include poor adherence, DTG monotherapy, advanced HIV and opportunistic infections.6 The family unit plays an important role in supporting children and adolescents. Decentralisation of care and other avenues of support such as community based family or youth clubs can also help to improve adherence.An evaluation of the ADVANCE (dolutegravir plus two different prodrugs of tenofovir to treat HIV) and NAMSAL  studies highlighted the impact of social and demographic factors on adherence even on DTG-based ART with younger age associated with reduced adherence.The value of VL monitoring and DR genotyping is crucial in detecting cases of resistance. With increasing transitioning to DTG, population surveillance for DTG resistance must be implemented. There is also a need for the availability of a wide repertoire of drug options that are effective, tolerable and cost-effective with child-friendly formulations.This report describes the first case of DTG resistance identified in the paediatric population in KwaZulu-Natal in a treatment-experienced adolescent. It highlights the impact of family and psychosocial issues on adherence, issues affecting adherence in children and paediatric population , and the need to closely monitor treatment-experienced patients with virologic failure, even on DTG-based regimens. The availability of the abacavir/3TC fixed-dose combination and DTG dispersible tablet will help address some of these issues and improve adherence.


Appointment keeping and self-report within 7-day or and 30-days recall periods are non-objective measures of antiretroviral treatment (ART) adherence. We assessed incidence of virological failure (VF), predictive performance and associations of these adherence measures with VF among adults on long-term ART. Data for persons initiated on ART between April 2004 and April 2005, enrolled in a long-term ART cohort at 10-years on ART (baseline) and followed until December 2021 was analyzed. VF was defined as two consecutives viral loads ≥1000 copies/ml at least within 3-months after enhanced adherence counselling. We estimated VF incidence using Kaplan-Meier and Cox-proportional hazards regression for associations between each adherence measure  and VF. The predictive performance of appointment keeping and self-reporting for identifying VF was assessed using receiver operating characteristic curves and reported as area under the curve (AUC). We included 900 of 1,000 participants without VF at baseline: median age was 47 years (Interquartile range: 41–51), 60% were women and 88% were virally suppressed. ART adherence was ≥95% for all three adherence measures. Twenty-one VF cases were observed with an incidence rate of 4.37 per 1000 person-years and incidence risk of 2.4% (95% CI: 1.6%-3.7%) over the 5-years of follow-up. Only 30-day self-report measure was associated with lower risk of VF, adjusted hazard ratio (aHR) = 0.14, 95% CI:0.05–0.37). Baseline CD4 count ≥200cells/ml was associated with lower VF for all adherence measures. The 30-day self-report measure demonstrated the highest predictive performance for VF (AUC = 0.751) compared to appointment keeping (AUC = 0.674), and 7-day self-report (AUC = 0.687). The incidence of virological failure in this study cohort was low. Whilst 30- day self-report was predictive, appointment keeping and 7-day self-reported adherence measures had low predictive performance in identifying VF. Viral load monitoring remains the gold standard for adherence monitoring and confirming HIV treatment response. The WHO public health approach for scaling up access to anti-retroviral therapy (ART) expanded following the availability of highly active anti-retroviral medication through standardized regimes and decentralized care in Low and Middle Income Countries (LMIC) . In thisIn 2020, an estimated 17 million people were on ART in sub-Saharan Africa (SSA) . The 201Undetectable equals Untransmittable (U = U) [Sustained optimal adherence to ART ensures virological suppression, reduction in HIV-related morbidity and mortality, and prevents onward transmission , 12, 13, (U = U) , 15. Dif (U = U) , 17. TheThus, this study aimed to describe the incidence of virological failure, compare the predictive performance of three ART adherence measures  and assess factors associated with virological failure among PWH on long-term first-line ART.This study was conducted at the HIV Centre of Excellence at the Infectious Diseases Institute (IDI) located in the Mulago Teaching Hospital in Kampala, the capital city of Uganda. The IDI clinic is a large out-patient clinic that currently serves over 8,000 PLWH in five municipalities in Kampala.This was a secondary analysis of a longitudinal cohort data of patients enrolled in the Long-Term ART cohort. The ART Long-Term cohort is an observational cohort of 1,000 patients who had been on ART for at least 10 years and were enrolled between May 2014 and September 2015 to be followed up for an additional 10 years . PLWH) wGeneral medical history, physical examination, adherence to ART, and prescription of drugs were performed at enrolment and all study visits. Follow-up visits were scheduled once a year for 10-years. In addition to study visits, the participants attended the general clinic every 3-months to pick up their ART and concomitant medications. Antiretroviral drugs were prescribed according to the WHO guidelines; PLWH with two consecutive viral loads >1000 copies/ml after enhanced adherence counselling were considered for treatment switch. Enhanced adherence counselling is a targeted counselling offered to PLWH on ART with non-suppressed viral load, done every month for at least 3-months before the next viral load test . At eachThe primary outcome was virological failure defined as two consecutive plasma HIV RNA viral load measurements ≥1000 copies/ml at least within 3-months after receiving enhanced adherence counselling following the first viral load measurement. The exposure was ART adherence assessed using 3 different measures: self-reported pill use in the last 7 days, self-reported pill use in the last 30 days, and appointment keeping. The 30-day and 7-day self-report of pill use ART adherence measure was assessed on a scale of 1–100 by asking the patient to recall the numbers of missed doses in the last 30-days and 7-days and then calculating, what percentage of ART doses were taken. Good adherence was determined as having a score ≥95%. Appointment keeping was defined as returning for a scheduled cohort clinic visit appointment or within a 7-day window after a missed clinic visit. Questions were assessed throughout the entire 5-year follow-up period. Additional co-variates included; age, sex, marital status, employment status, HIV disclosure status, household level of income, and body mass index.We extracted cohort data from 10 to 15 years on ART follow-up (or enrollment in the cohort and five years of follow-up). When the required data were missing, patient charts were retrieved and reviewed to supplement the data in the databases. We extracted clinical data including ART start dates and regimens, socio-demographics at cohort enrolment, behavioral data, CD4 cell counts, plasma HIV viral load measurements for the follow-up period using Roche Cobas AmpliPrep/Cobas Taqman platform using viral load 20 copies/ml as a cut of. We also extracted data on deaths, transferred out, and lost to follow-up.Statistical analysis was performed using STATA 16.1 . We described cohort participants using frequencies and percentages for categorical variables, continuous variables using means and standard deviations and medians and interquartile ranges. Adherence measures were described using frequency and percentages across calendar year. Kaplan-Meier methods were used to estimate incidence risk and incidence rate of virological failure. Associations between virological failure and ART adherence was examined using Cox-proportional hazards regression (Cox-PH) models. ART adherence measures were entered into the model as time-dependent covariates measured at annual cohort visits. The Schoenfeld residuals test was used to assess for violation of the Cox–PH assumption. Two sensitivity analyses were performed, by refitting the model when: 1) missing values on covariates were imputed by multiple imputation using chained equations (MICE), 2) considering all censored patients i.e., deaths and losses to follow-up as virological failure (worst-case scenario), Performance of ART adherence measures—appointment keeping, 30-days and 7-days self-report of pill use for predicting virological failure was evaluated using receiver operating characteristic curve analysis. All hypothesis tests were performed as 2-tailed tests at a 5% significance level.This study was approved by the Infectious Diseases Institute Research Ethics Committee  and the Uganda National Council for Science and Technology . The IDIREC committee granted a waiver of informed consent since secondary data were retrieved and analysed..We retrieved data for 1,000 PLWH adults enrolled in the long-term ART cohort who started ART between April 2004 and April 2005 and were followed up until December 2021. Of the 1,000 participants, 100 (10%) had a viral load (VL) >1000 copies/ml documented at cohort enrolment and were therefore excluded from the study. Nine hundred participants were included in the analysis, of whom 10 had transferred to other health facilities, 45 were lost to follow-up and 41 had died before reaching 15 years on ART .Of the 900 cohort participants analyzed, at cohort enrollment: the median age was 46 years (IQR 41–51); 59.8% were females, 82.1% were employed, 43.9% lived <1 US dollar per day, median body mass index (BMI) was 22.4 (IQR 19.8–25.4), 51.3% were married or cohabiting, and 88.4% had viral load <50 copies/ml (10 years on ART), ( ART adherence was very high (≥95%) over the 7-calendar years studied on all adherence measures, except appointment keeping declined in 2019 and 2020  .The virological failure incidence rate was 4.37 (95% CI: 2.85–6.70) per 1000 person-years and the probability of virological failure was 2.4%, (95% CI: 1.6% - 3.7%) over 15 years, . There was no significant association with 7-day self-reported or appointment keeping measures .In the receiver operating characteristics curve (ROC) analysis for predictivity ability of adherence measures for virological failure, 30-day self-report best predicted virological failure  versus appointment keeping  and 7-day self-report   which found a low rate of virological failure among adult PLWH on first-line antiretroviral therapy . Our stuWe found that baseline CD4 cells ≥200 cells/ml was associated with lower risk of developing virological failure in models using each of the three adherences measures. Our finding is similar to several studies both in developed and LMIC that have reported low baseline CD4 count have increased risk of developing virological failure . This fiWe also found that all the three adherence measures had low predictive performance in identifying virological failure. However, the 30-day self-report adherence measure was most able to predict virological failure. Our finding that 30-day self-report predicted virological failure is consistent with a study by Minyi et al., 2008  who founundetectable equals to untransmissible (U = U). As we disseminate and implement the UNAIDS policy, programs should integrate objective methods of measuring adherence like medication event monitoring systems, and biologic measures like point of care tenofovir testing that best predict virological failure.To improve ART adherence, HIV care programs in LMIC should continue to educate people living with HIV on the importance of reporting accurate and consistent ART adherence, keeping dosing schedules, and explaining adverse effects. National ART guidelines should pay particular attention to monitoring virological failure and supporting ART adherence among persons who initiate ART with lower CD4 count. Monitoring viral load helps identify PLWH on ART who have sustained long-term viral load suppression and is crucial in HIV prevention efforts given that national programs are promoting the UNAIDS slogan of The key strength of our study is the prospective data collection design among long-term ART persons, large sample size, long duration of follow-up, and objective ascertainment of virological failure. However, our study has limitations. These findings may not be generalizable because persons were from an HIV centre of excellence, which may not be representative for smaller centres or primary care settings, social desirability bias, immortal time bias could have affected the study findings. Also, this is a non-randomized comparison and is subject to unmeasured confounding. Twenty percent of viral load data were missing but we used multiple imputation in the sensitivity analysis.The incidence of virological failure among PWA on long-term ART in this cohort study was low. Unlike the 30-day self-report, appointment keeping and 7-day self-reported ART adherence measures had low predictive performance in identifying virological failure. Routine plasma viral load monitoring remains the gold standard for adherence monitoring and confirming HIV treatment response.S1 FileTable A: Association of ART adherence and virologic failure, adjusting for baseline patients’ characteristics –worst-case scenario.(DOCX)Click here for additional data file.S1 Dataset(XLSX)Click here for additional data file.


Viral load assessment for people living with HIV is key for monitoring treatment and achieving the 95-95-95. In this study, we aimed to assess the degree of viral suppression at different thresholds and treatment duration after the introduction of dolutegravir-based therapy in ten public hospitals in Sierra Leone.We used a cross-sectional study design to recruits patients aged 18 years or older between August 2022 and January 2023. Statistical analyses were performed using R-software. Logistic regression was used to assess factors independently associated with viral suppression. The level of significance was set at P < 0.05.Of the 2,253 patients recruited, 1,720 (76%) were women and 1,705 (76%) were receiving a fixed dose combination of tenofovir, lamivudine and dolutegravir. The median age and duration of anti-retroviral therapy (ART) was 36.0  years and 40.9  months, respectively. Using a threshold of HIV RNA < 1000 copies/mL, 1,715 (88.4%) patients on ART for more than 6 months were virally suppressed. Viral suppression rates were higher with dolutegravir-based  than efavirenz-based  ART. HIV RNA was < 200 copies/mL in 1,643 (84.6%) patients or < 50 copies/mL in 1,487 (76.6%) patients or between 50 and 999 copies/mL in 228 (11.7%) patients. Viral suppression rates at different ART durations (months) were as follows: 84.2% (≤ 3), 88.8% (4–6), 90.9% (6–12), and 88.1% (> 12). Viral suppression rates were higher for patients aged 40 or older . Men had 49% lower odds of viral suppression than women .We report a viral suppression rate of 88.4% among patients on treatment for at least 6 months, with higher rate of suppression with dolutegravir than efavirenz. Factors associated with virological suppression were age and gender, emphasizing the need for innovative differentiated ART delivery models to optimize viral suppression and achieve the 95% target. A fast-track strategy to end the HIV epidemic calls for low and middle-income countries to adopt the Joint United Nations Agency on HIV/AIDS (UNAIDS) 95-95-95 global target; 95% of people living with HIV (PLHIV) should know their status, 95% of those who know their status should receive antiretroviral treatment (ART) and 95% of those in care should have a suppressed viral load by 2030. The World Health Organization (WHO) recommends viral load assessment for all PLHIV as a key intervention for treatment monitoring in PLHIV and achieving the third 95 . These oSierra Leone is a low HIV prevalence setting with a national seroprevalence of 1.7% in 2019. However, the country faces several challenges in its response to the HIV epidemic, including a high prevalence of advanced HIV diseases, late-stage diagnosis, and opportunistic fungal and non-fungal infections . Prior tSince the introduction of regimen transition, viral load assessment has been challenging, and it is unclear what effect dolutegravir-based therapy has on viral suppression. Understanding the effect of dolutegravir-based therapy on different HIV viral thresholds at different treatment durations has important policy implications, as it provides data to understand the implications of lowering the viral threshold on the scarce resources of low-income countries . In thisWe used a cross-sectional study design to collect primary data from adult patients aged 18 years or older between August 2022 and January 2023.Sierra Leone is a low-income country in West Africa with five geographical regions, such as the Western Area and North, East, South and Northwestern regions with a population of 7 million in 2015. About 22% (1.5 million) of its population live in the Western Area . The heaAt the time of the study, there were 439 sites reporting data to the Sierra Leone National AIDS Control Program, of which 215 sites accounted for just 2% of the more than 58,000 PLHIV receiving ART by April 2022. There are 72 large sites where at least 100 PLHIV are receiving ART, accounting for 86% of all PLHIV receiving ART in the country.We selected health facilities where at least 800 PLHIV were receiving treatment by April 30, 2022 at a time when the study is being planned and conducted the study in 10 of these public health facilities. The health facilities and their PLHIV populations included Connaught Hospital , Waterloo Community Health Center , Rokupa Government Hospital , Jenner Wright Clinic , Princess Christian Maternity Hospital , Lumley Government Hospital (988) and 34 Military Hospital (804) in the Western Area, Bo Government Hospital  in the South, Kenema Government Hospital  in the East, and Makeni Government Hospital  in the North. Two centers in the East  and two in the Western Area (Wellness Clinic and UMC Urban Center) with at least 800 PLHIV were excluded because of the distance or delay in obtaining consent to conduct the study. Connaught Hospital, Kenema Government Hospital, Makeni Government Hospital, Bo Government Hospital and 34 Military Hospital are tertiary/regional hospitals, while Rokupa and Lumley Government Hospitals are secondary hospitals. The remaining two facilities  are primary health facilities.After providing written informed consent, we recruited participants non-randomly as they presented to the different health facilities between August 2022 and January 2023. Demographic and HIV information were collected from patients using a standardized data collection form and cross-checked with clinical records.Approximately 10 mL of venous blood was collected aseptically in EDTA vacutainer tubes and analyzed centrally at the Infectious Disease Prevention Center of the 34 Military Hospital in Freetown, Sierra Leone. The plasma was separated by centrifugation and stored at -80 °C until use. The HIV RNA was extracted using the TGuide S32 Magnetic Viral DNA/RNA Kit  and the HIV viral load was determined by HIV-RNA Quantification Kit  according to the manufacturer’s instructions. Amplification of HIV-1 RNA and standards were detected by the probes labeled with FAM channel and Select VIC/HEX/YELLOW channel to detect HIV Internal Control. HIV-1 RNA amplification and quantification were performed with the CFX96 real-time PCR detection system . Thermal cycling conditions used were 95 °C for 1 min for pre-denaturation and enzyme activation; 60 °C for 30 min reverse transcription; 95 °C for 1 min for cDNA pre-denaturation; 95 °C for 15 s for denaturation, and 58 °C for 30 s for annealing, extension and fluorescence collection followed by 45 cycles. The threshold for HIV RNA detection is 15 copies/mL.Statistical analyses were performed using the R-software version 4.2.3 . Baseline characteristics were summarized using frequencies and medians. The differences in patient characteristics were assessed using Pearson’s Chi-squared test, Fisher’s exact test and Kruskal-Wallis H test. Multivariable binary logistic regression analysis was used to assess factors independently associated with viral suppression. The level of significance was P < 0.05.Over the course of the study, 2,253 patients were recruited. Their median age was 36.0  years. More than a third were under 30 years  and nearly half were married . Most were women , had been on ART for more than 24 months  and were receiving a fixed dose combination of tenofovir, lamivudine and dolutegravir   had an HIV RNA level < 1000 copies/mL, regardless of the duration of ART Table . Using aViral suppression rates were 84.2% for those who had been on ART for 3 months or less, 88.8% for those who had been on ART for 4 to 6 months, and 90.9% for those who had been on ART for 6 to 12 months. The viral suppression rate was 88.1% in patients receiving ART for more than 12 months ’ and emphasizes the benefits of early HIV diagnosis and treatment . A viralFactors associated with suppressed viral load < 1000 copies/mL are older age, female sex and level of service delivery. Similar findings were reported in adolescents in Uganda, where age and duration of antiviral therapy are associated with virologic suppression . In EthiOur study has limitations. First, the study did not assess the status of adherence to ART in the different facilities and therefore has no information on the impact of the treatment interruptions on HIV viral suppression. HIV drug resistance and baseline CD4 count may be important predictors of viral suppression but were not assessed due to insufficient resources. The study was only conducted at large sites, and the results may differ from smaller facilities, where staff may be less well trained and care may be different. Finally, we plan to recruit 20% of PLHIV from each of the 10 sites between August 2022 and January 2023, but this plan was not achieved in Waterloo Community Health Center and Kenema, Bo and Makeni Government Hospitals due to the slow flow of patients. Thus, of the 19,901 PLHIV receiving ART in all these facilities as of April 30, 2022, only 2,253 (11.3%) were enrolled.Nonetheless, this study provides national insights into the state of HIV viral suppression and proffer actionable recommendations on viral load management in Sierra Leone and other countries facing similar challenges.We report a viral suppression rate of 88.4% among PLHIV treated with ART for at least 6 months, with higher rate of suppression in dolutegravir than efavirenz-based therapy. Common factors associated with virological suppression were age and gender, emphasizing the need for innovative differentiated ART service delivery models to optimize viral suppression and achieve the national target of 95%.


Monitoring of HIV drug resistance (HIVDR) remains critical for ensuring countries attain and sustain the global goals for ending HIV as a public health threat by 2030. On an individual patient level, drug resistance results assist in ensuring unnecessary treatment switches are avoided and subsequent regimens are tailored on a case-by-case basis, should resistance be detected. Although there is a disparity in access to HIVDR testing in high-income countries compared to low- and middle-income countries (LMICS), more LMICs have now included HIVDR testing for individual patient management in some groups of patients. In this review, we describe different strategies for surveillance as well as where HIVDR testing can be implemented for individual patient management. In addition, we briefly review available technologies for HIVDR testing in LMICs, including Sanger sequencing, next-generation sequencing, and some point-of-care options. Finally, we describe how South Africa has implemented HIVDR testing in the public sector. Sustained high levels of community HIV viral load (VL) suppression are critical to achieving the WHO/UNAIDS 95-95-95 target of eliminating AIDS as a public health threat by 2030 . HoweverHIV drug resistance testing is recommended for individual patient management in many resource-rich settings ,4,5, butIn countries where routine resistance testing is not feasible, it is advised to conduct regular surveys to monitor: pretreatment drug resistance (PDR) in adults initiating or re-initiating first-line ART, PDR in infants newly diagnosed with HIV, acquired drug resistance (ADR) in adults, adolescents, and children with virologic failure (VF) on ART, and HIV-diagnosed pre-exposure prophylaxis (PREP) recipients ,8.Here, we review the impact of HIVDR testing on treatment guidelines and individual patient management in LMICs, as well as what technologies are available and required in the near future to address the need for HIVDR testing with the growing number of patients on ART and changing regimens.Global and national ART guidelines rely on the availability of reliable national data, including treatment outcomes and HIVDR prevalence trends. Now that routine VL testing is widely available, programmatic data can often be used to assess the responses to ARV regimens at a population level. However, population-level implementation of HIVDR testing is limited. Therefore, one has to rely on nationally representative surveillance data instead.Over the years, the WHO has published a number of surveillance strategies, targeting different populations and providing tools to calculate target sample sizes for each of the methods.For the assessment of PDR, a cross-sectional clinic-based survey approach is recommended . In the The protocol to assess HIVDR in children less than 18 months of age recommends the use of remnant dried blood spots (DBS) from early infant diagnosis (EID) testing over a period of 12 months . In thisThree different methods were proposed to assess the acquired HIVDR, which can be applied to adults, adolescents, and children.Strategy one: For countries with at least 60% VL testing coverage, a nationally representative laboratory-based method is recommended . It targStrategy two: For countries with inadequate VL testing coverage (<60%), a nationally representative clinic-based method is suggested . This apStrategy three: Should both of these approaches not be feasible, WHO has proposed a sentinel laboratory-based method targeting patients on DTG-based ART . This suOutcomes of PDR surveys provide information to select nationally recommended first-line ART regimens, especially in countries where baseline HIVDR testing is not available. During the initial ART roll-out phase in LMICs 2004–2010), HIVDR levels among patients initiating treatment remained relatively low, but were estimated to increase at a rate of 29% per year in East Africa and 14% per year in southern Africa . More re004–2010,Although the implementation of PMTCT regimens has greatly reduced the vertical transmission of HIV-1, at least half of the vertical transmissions involved drug-resistant viruses ,16,17. BSurveillance of ADR provides critical information needed to assess the performance of ART programs in achieving VL suppression targets. The pooled analysis of VL suppression in the African region between 2014 and 2020 was 94% in patients receiving NNRTIs ,26,27. MSurveillance data on resistance profiles in patients failing PI-based ART is scarcer and was not included in the WHO resistance reports. However, a systematic review and meta-analysis of studies reporting outcomes of PI-based second-line ART in sub-Saharan Africa showed viral suppression rates of 69% and 58% at 48 and 96 weeks, respectively. Major protease resistance mutations were observed in 17% of those failing second-line PI-based ART. This prevalence, however, increased with the duration of second-line ART . In NamiGiven the recent large-scale roll-out of DTG, surveillance data on DTG resistance are still uncommon. Only four countries reported viral suppression rates for patients on DTG-based ART in the latest WHO HIVDR report . Viral sIn high-income countries where individualized treatment is available, HIVDR testing is recommended at the time of VF ,4,5. AltA recent meta-analysis of studies assessing the efficacy, safety, and tolerability of DTG in first-line ART showed that none of the patients developed DTG resistance, indicating most failures are due to suboptimal treatment adherence . AlthougSwitching patients from a NNRTI-based ART to tenofovir-lamivudine-dolutegravir (TLD) has proven to be a successful strategy, with high levels of viral suppression obtained ,40,41,42Current technologies for HIVDR genotypic testing include Sanger sequencing, next-generation sequencing (NGS), and point mutation assays (PMAs). Although not yet commercially available, efforts are being made to develop point-of-care (POC) HIVDR tests.https://hivdb.stanford.edu/ (accessed on 28 June 2023)), Geno2Pheno (https://genafor.org/services.php (accessed on 28 June 2023)), ANRS (https://hivfrenchresistance.org/ (accessed on 28 June 2023)) and Rega (https://rega.kuleuven.be/cev/avd/software (accessed on 28 June 2023)).Traditionally, most laboratories use Sanger sequencing to assess resistance in viral populations. This target-specific PCR-based technique produces a single consensus sequence of all virus variants present in at least 15–25% of the viral population in a specimen. When the equipment is accessible, Sanger sequencing is relatively simple to perform, and is relatively inexpensive. Several commercial and laboratory-developed methods are available . CommercNext-generation sequencing (NGS) performs high-throughput, massive parallel sequencing, which enables a high sequence depth for the identification of low-abundance variants. If sufficient sequence coverage is ensured, NGS can detect variants present in 1% of the viral population in a specimen, provided a minimum coverage of 1000 sequences per base is used and the HIV RNA is at least 1000 copies/mL. Below this threshold, it is extremely difficult to distinguish true low-frequency variants from PCR errors ,47. The Apart from increased sequence depth, NGS can be leveraged to increase the coverage across the HIV genome to include protease, reverse transcriptase and integrase, or even perform whole genome sequencing at minimal additional cost. The NGS field is currently dominated by Illumina and Ion Torrent platforms, while other players, such as Oxford Nanopore Technologies, have recently joined the competition. Similar to Sanger sequencing, there are now commercial and in-house methods available to perform HIVDR testing using multiple platforms . A varieAlthough NGS is cheaper than Sanger, when comparing the cost per base, savings can only be realized when multiple samples are pooled in a single run. This practice would make NGS suitable and affordable for surveillance activities where large batches of samples are available for testing. On the other hand, when considering implementing NGS for individual patient management, one has to consider the trade-off between cost and turn-around time. In countries with large HIV epidemics, this could be overcome by centralizing testing.The availability of point-of-care (POC) assays could offer a decentralized testing approach. Most POC assays, though not yet commercially available, are designed on the principle of detecting a handful of clinically relevant mutations rather than providing a comprehensive overview of all mutations present.Point-of-care, or near POC assays, would reduce the need for laboratory equipment to a minimum while shortening the time to results, with the trade-off of missing the complete HIV genetic sequence information. Recent advances in the development of POC assays for HIVDR testing were reviewed by Chua et al. . One tecAnother interesting technology is the pan-degenerate amplification and adaptation (PANDAA) technology, which is based on quantitative PCR and uses extremely degenerate primers that target specific mutation sites and can cope with high HIV diversity next to the mutation site . This teHowever, no data has been presented yet on the detection of integrase strand transfer inhibitor mutations for either technology.In South Africa, which has the largest ART program globally, a combination of HIVDR testing strategies is used for individual patient management and surveillance. The National Health Laboratory Service (NHLS) provides a laboratory network covering all patients treated in the public sector, which equates to approximately 80% of the population. In 2013, two laboratories offered HIVDR tests for individual patient management. Three additional laboratories were included in this network from 2015 onward to ensure adequate geographical coverage within the country. Virologists and laboratory personnel from each of the five laboratories established an HIVDR committee with the aim of streamlining technical and quality assurance processes and offering support when technical or logistical challenges are encountered. Since 2013, the South African treatment guidelines have recommended HIVDR testing for patients failing PI-based ART with confirmed VF and at least two years of exposure to PIs ,56,57,58In 2022, the NHLS performed approximately 4500 HIVDR tests for individual patient management, of which most were for patients failing PI-based ART and approximately 5% included integrase testing. When PI or InSTI resistance is identified, a full treatment history is submitted to the Third Line Review Committee via the National Department of Health for consideration. Once consensus is reached, a treatment decision is conveyed to the local facility, and if third-line ART (TLART) is indicated, the drugs are dispatched to the facility on a named patient basis. The South African TLART access program is the first and largest of its kind in the public sector and has over 4000 patients on the database as of August 2022.The National Institute for Communicable Diseases (NICD) and the Human Sciences Research Council (HSRC) conduct the South African HIV Prevalence, Incidence, Behavior and Communication Household Surveys (SABSSM). The most recent SABSSM survey was conducted in 2017, prior to the DTG rollout. Drug-level testing was used as a proxy for adherence. HIVDR was detected in 27% of patients with VF, but this increased to 75% among those who had discontinued treatment . LaboratAddressing and monitoring HIVDR remains an essential component in the fight against HIV/AIDS. Despite the inequality in access to HIVDR testing in different regions of the world, more LMICs now have access to the technologies to perform HIVDR testing for surveillance purposes or individual patient management. In this review, we describe the different methodologies recommended by WHO for HIVDR surveillance and the impact these results have on treatment guidelines. Although these surveillance activities are commendable, they remain a resource-consuming exercise that many countries cannot afford to perform on a regular basis. In addition, the WHO HIV drug resistance report often includes relatively old data due to delays in the sharing of data, data curation, and meta-analysis. As part of the response to the SARS-CoV-2 pandemic, sequencing technologies now have a larger footprint in LMICs. However, reagent costs, investment in continuous maintenance of equipment, and sustained technical skills training remain limited in some regions. Next, we presented a nonexhaustive summary of available technologies for HIVDR testing in LMICs, including Sanger sequencing, next-generation sequencing, and some point-of-care options. We acknowledge that we did not perform a systematic review of the literature, which might have led to the omission of some of the newer technologies under development.In order to end the HIV epidemic, we require universal access to treatment and sustained viral suppression at the community level. HIVDR is a major barrier to achieving viral suppression; therefore, the availability of HIVDR testing for individual patient monitoring and adequate surveillance programs are pivotal to ensuring ART efficacy for both individual patients and public healthcare. Continuous investment in the development of newer, more accessible technologies and capacity building in LMICs is required. Skills development for laboratory staff, improved technical support by suppliers, and cost negotiations for reagents should be prioritized.


Following reports of anti-retroviral therapy (ART) experienced Ugandan people living with HIV (PLHIV) presenting with diabetic ketoacidosis weeks to months following a switch to dolutegravir (DTG), the Uganda Ministry of Health recommended withholding DTG in both ART naïve and experienced PLHIV with diabetes mellitus (T2DM), as well as 3-monthly blood glucose monitoring for patients with T2DM risk factors. We sought to determine if the risk of T2DM is indeed heightened in nondiabetic ART naïve Ugandan PLHIV over the first 48 weeks on DTG.Between January and October 2021, 243 PLHIV without T2DM were initiated on DTG based ART for 48 weeks. Two-hour oral glucose tolerance tests (2-h OGTT) were performed at baseline, 12, and 36 weeks; fasting blood glucose (FBG) was measured at 24 and 48 weeks. The primary outcome was the incidence of T2DM. Secondary outcomes included: incidence of pre-Diabetes Mellitus (pre-DM), median change in FBG from baseline to week 48 and 2-h blood glucose (2hBG) from baseline to week 36. Linear regression models were used to determine adjusted differences in FBG and 2hBG from baseline to weeks 48 and 36 respectively.p-value (p) = 0.005] and significant reduction in 2hBG  at week 36. A high CD4 count and increased waist circumference were associated with 2hBG increase at week 36.The incidence of T2DM was 4 cases per 1000 PY (1/243) and pre-DM, 240 cases per 1000 person years (PY) (54/243). There was a significant increase in FBG from baseline to week 48 [median change from baseline (FBG): 3.6 mg/dl, interquartile range (IQR): − 3.6, 7.2, We demonstrated a low incidence of T2DM in Ugandan ART-naïve patients receiving DTG. We also demonstrated that longitudinal changes in BG were independent of conventional risk factors of T2DM in the first 48 weeks of therapy. Restricting the use of dolutegravir in Ugandan ART naïve patients perceived to be high risk for diabetes mellitus may be unwarranted.The online version contains supplementary material available at 10.1186/s12981-023-00510-6. In the early anti-retroviral therapy (ART) era, nucleoside reverse transcriptase inhibitors (NRTIs) including didanosine, zidovudine, stavudine, or lamivudine were coupled with non-nucleoside reverse transcriptase inhibitors (NNRTIs) , 2. SomeAfter multiple countries reported primary resistance to NNRTIs above the recommended 10% threshold, the World Health Organisation (WHO) recommended the use of dolutegravir (DTG), a later generation integrase inhibitor as part of first and second line therapy , 9. Mult2 and history of hypertension at baseline  of the patients were in HIV clinical stage 1 with 35 (14%), 14 (5.8%), 2 (0.8%) being in HIV clinical stages 2, 3 and 4 respectively. Nine (3.7%) patients had an established diagnosis of active tuberculosis diagnosed at enrollment into HIV care. Overall, 61% had a normal BMI, 23.5% were overweight, 10% underweight and 5.8% obese. Most (81%) of the patients self-reported as meeting WHO recommendations on physical activity for health. The median serum creatine, fasting LDL, fasting HDL and fasting total cholesterol at baseline were: 0.83 mg/dl, 78.1 mg/dl, 31.7 mg/dl and 136.1 mg/dl respectively. At 24 weeks of follow up, 238 patients (99.6%) had viral suppression  were female. The median age of the participants was 31 years (IQR:27–38) with a median CD4 cell count of 318 cells/mmThere were not significant baseline study characteristic differences between patients who completed 48 weeks of follow up and those who did not . The incidence of pre-DM was 240 cases per 1000 person-years (PY) (54/243). Of the 54 patients diagnosed with pre-DM, 40 patients had a subsequent visit after pre-DM diagnosis (diagnosed between 12 and 36 weeks) and majority 32/40 (80%) reverted to a normal blood glucose state within the next 12 weeks.p = 0.005) . Thereafter, was a slight increase to 36 weeks. Despite the rise in 2hBG from week 12 to week 36, the median 2hBG at 36 weeks was significantly lower than at baseline   = 0.045]. Additionally, having a waist circumference corresponding to increased risk of cardiometabolic complications at baseline was associated with an increase in 2hBG at week 36 . There were no factors associated with increase in FBG at 48 weeks : 0.0002–0.021, In this study, we determined that over 48 weeks of DTG-based ART, the incidence of T2DM was low [4 cases per 1000 PY (0.4%)] with the majority of the patients diagnosed with pre-DM having transient increase in FBG/2hBG.The incidence rate reported in our study is similar to the rates reported in the safety data of two DTG efficacy randomized controlled trials (RCTs) with ART naïve PLHIV in Cameroon and South Africa. The ADVANCE (96-week follow up) and NAMSAL (48-week follow up) studies reported incidences of 0.9% and 0.3% with controls on NNRTIs having 0.3% and 0% respectively , 39. PatFrom the Makerere University Infectious Diseases Institute report, over 12 months the reported incidence of T2DM was 4·7 per 1000 PY in the case (ART naïve and exposed PLHIV started on DTG) versus 0·32 per 1000 PY in the control group (PLHIV on other first line regimens without DTG). Almost all (15/16) of the T2DM cases were ART exposed before the switch to DTG. One limitation of that report was that the diabetes statuses of patients were not known at the time of ART switch and the cases reported were the overtly symptomatic DKA patients hence a possible underestimation of the burden of hyperglycemia. The possible underestimation of the burden of incident T2DM may partially have informed the MoH guidelines. In contrast, our study recruited only patients free of T2DM at baseline with prospective proactive screening for T2DM irrespective of symptoms using a strict criterion for T2DM diagnosis hence may better represent the incidence estimates in the naïve group, which is low.In our study, there was a drop in both FBG and 2hBG in the first 12 weeks of follow-up, followed by a consistent increase up to 48 weeks. Despite the 48-week FBG being significantly higher than at baseline, the 2hBG at 36 weeks was less. The trajectories suggested a steady increase in BG after 12 weeks suggesting that with longer follow up there could be a consistent significant increase in both fasting and 2hBG. Multiple factors interact in regulating blood glucose. These include: basal insulin secretion, post prandial insulin secretion, hepatic gluconeogenesis, insulin resistance and insulin clearance –44. SystFrom our analysis, both FBG and 2hBG changes between baseline to 48 weeks were not associated to conventional risk factors for diabetes including: baseline BMI, physical inactivity and increase in BMI over the follow up period, apart from increased waist circumference which was associated with increase in 2hBG at 36 weeks. In the Ugandan HIV treatment guidelines, these are the parameters used to stratify T2DM risk to determine which patients to screen for T2DM every 3 months . AdditioOur study had various limitations. We did not have a comparator group hence we could only describe the natural history of glucose changes in our cohort but not conclusively attribute the changes in BG to exposure to dolutegravir. It was a single center study in an urban area hence the results may not be generalizable to the general PLHIV population in Uganda. Much as the patients that were excluded during the study did not have significantly different clinical and demographic characteristics compared to those that completed the study, the dropout rate was high which could lead to under- or over-estimation of our end point depending on the outcome of the dropout participants. At enrollment, very sick patients were excluded, the same group of patients that may have more heightened systemic inflammation with different glucose metabolism trends on introduction of ART. Despite the limitations, our study had a clearly defined metabolic end point with a strict and sensitive criterion for the diagnosis of T2DM making our results reliable . We screWe demonstrated that the incidence of T2DM in Ugandan ART naïve patients over 48 weeks of DTG anchored ART is low. Nevertheless, there was a trend towards rising blood glucose after week 12. We also demonstrated that longitudinal changes in BG were independent of conventional risk factors of T2DM in the first 48 weeks of therapy. Restrictive use of DTG in Ugandan PLHIV perceived to have a high risk for T2DM may be uncalled for.Additional file 1. Table S1: Sensitivity analysis comparing baseline characteristics of participants who dropped out and those who did not.


This study examined the prevalence and factors associated with detectable viremia, as well as clinical outcomes among people with HIV (PWH) receiving antiretroviral therapy (ART) who initially achieved viral suppression in 2 southern states in Nigeria.The retrospective cohort study used data from the electronic medical records of 96 comprehensive ART centers. PWH were followed up who achieved viral suppression  upon starting ART based on the first VL test. We examined the presence of detectable viremia in follow-up VL results, graded by the absolute VL count from the second and third consecutive VL tests as follows: transient viremia , persistent viremia , and virologic failure . We analyzed demographic and clinical factors associated with detectable viremia using logistic regression analysis on Stata 14.P < .001), being 0 to 14 years of age (P < .001), and not being enrolled in a differentiated service delivery model (P < .001) were significantly associated with detectable viremia.Overall, 15 050 PWH had achieved viral suppression following ART initiation . On follow-up, 3101 (20.6%) had a viremic event: 11.6%, transient viremia; 8.8%, persistent viremia; 0.2%, virologic failure. Shorter duration of ART (Our study shows that people who initially attain vial suppression upon starting ART remain at risk of detectable viremia. This article provides further evidence for the need for enhanced monitoring and management of people living with HIV even after they have achieved viral suppression. It highlights priority subgroups for enhanced care, including those not enrolled in a differentiated service delivery model, those aged 0 to 14 years, and people living with HIV who have been undergoing antiretroviral therapy for a shorter period. The main goal for antiretroviral therapy (ART) is to achieve viral suppression at the individual and population levels . GloballSustained ART maintains maximal suppression of the viral load , restores or preserves immunologic function, improves quality of life, and reduces HIV-related morbidity and mortality , 5. MainPWH who achieve undetectable VL levels may subsequently experience transient or persistent viremia . PersistThis study describes the prevalence of detectable viremia among people who initially achieve viral suppression. We also describe demographic and clinical factors associated with detectable viremia as well as their clinical outcomes.We conducted a secondary analysis of program data for PWH receiving ART from 96 health facilities in Akwa Ibom and Cross River States in Nigeria. We analyzed those who had an undetectable VL (≤50 copies/mL) as of December 2019 and 2 follow-up VL results  [The study was conducted in 2 states in Nigeria (Akwa Ibom and Cross River States), where the United States Agency for International Development funded the EpiC project (Meeting Targets and Maintaining Epidemic Control) to provide comprehensive HIV care and treatment services in 154 health facilities  and 101 community pharmacies. The project deployed 67 community ART management teams  “Compos.ART services, including treatment monitoring, was provided according to the “National Guideline for HIV Prevention, Treatment and Care” . ClientsData for this study were obtained from the electronic medical records of the Lafiya Management Information System in each health facility . AbstracART regimen (first line only) was classified into the following categories: HIV integrase inhibitors (tenofovir/lamivudine/dolutegravir and abacavir/lamivudine/dolutegravir), protease inhibitors (abacavir/lamivudine/lopinavir/ritonavir and tenofovir/lamivudine/lopinavir/ritonavir), and nonnucleoside reverse transcriptase inhibitors (tenofovir/lamivudine/efavirenz and tenofovir/lamivudine/nevirapine).Maintained durable viral suppression: the 2 follow-up VL results were ≤50 copies/mL.The date of the initial VL test following ART initiation was taken as the baseline. We categorized virologic outcomes after the first VL test (first follow-up test) based on the results of the 2 follow-up VL tests:Transient viremia: the second follow-up VL was 51 to 999 copies/mL, but the third result was ≤50 copies/mL.Persistent viremia: the second follow-up VL was either 51 to 999 or ≥1000 copies/mL, but the third result was >50 copies/mL.Virologic failure: the second and third follow-up VL results were >1000 copies/mL .PWH reviewed were classified at follow-up as such: active (if the client maintained treatment until the end of the review period in March 2023), deceased, stopped treatment, or lost to follow-up (if the client had not picked up an ART refill for ≥28 days from the last expected refill appointment within the review period).P values <.05 were statistically significant.Descriptive statistics were used to analyze categorical variables and summarized by frequency, while continuous skewed variables were summarized as median and IQR. VL events  were summarized descriptively to determine distribution by the various client demographic and clinical characteristics. We used logistic regression analysis to calculate unadjusted and multivariable-adjusted odds ratios and 95% CIs to determine factors associated with viremia. All Chi-square statistics was then used to compare treatment outcome by ranges of VL. We dichotomized treatment outcome into “active” vs “treatment interruption.” All statistical analyses were performed in Stata 15 (StataCorp LLC).Permission to conduct a secondary analysis on these routine data was provided by Family Health International's Office of International Research Ethics . The ethics committee waived the requirement for informed consent by participants, as VL testing is part of the Ministry of Health’s routine standard of care for monitoring HIV treatment response. Also, data from the electronic medical records used for analysis in the study were fully anonymized, as clients’ names were excluded in the abstraction of data.Of the 25 511 records of children and adults active on ART as of December 2019, 17 052 records had at least 3 VL results within the period of review. An overall 2002 records were excluded because they contained VL results <6 months apart (VL after enhanced adherence counseling after a VL ≥1000 copies/mL). A total of 15 050 (64%) records categorized as unsuppressed were included in this analysis, .The sociodemographic and baseline clinical characteristics of the 15 050 clients are shown in In multivariate analysis, predictors of having any viremia included not being enrolled in a DSD model , receiving ART for <3 years , and being 0 to 14 years of age  maintained durable viral suppression, but 20.4% experienced viremic events during follow-up VL testing, with 0.9% ultimately developing virologic failure. Our results agree with findings from a study in Zimbabwe , 24, whiIn contrast to studies reporting lower numbers of clients with durable viral suppression (34%–57%) , 25, neaDetectable viremia is associated with poor adherence ; other fWhereas Young et al reported higher virologic failure rates among PWH who experienced low-level viremia in a Swiss HIV cohort study , <1% of Our study had some limitations. First, we did not consider the effect of ART adherence on the incidence of viremia, owing to the lack of good objective measures of clients’ ART adherence. Also, we used routinely collected data, with the attendant risk of missingness, and only fewer variables collected. Despite these limitations, the large sample size strengthens our findings. To our knowledge, this is the first study in Nigeria to stratify VL by finer categories, which allowed study factors associated with any of these outcomes.In this study, the prevalence, treatment outcome, and risk factors associated with detectable viremia were investigated in a cohort of PWH undergoing ART in 2 southern states in Nigeria. Data from this study provide evidence for the need for ongoing adherence support even among clients who attain virologic suppression upon starting ART. According to our data, nearly 20% of PWH who initially attain virologic suppression subsequently developed a virologic event, and a few went on to develop virologic failure. Findings from this study point to priority subgroups for enhanced care, including those not enrolled in a DSD model and those with a shorter duration of ART.


Doppler data showed a trend in increase of IMT > 1 in the PI-based group, which appeared statistically significant for the section of the left common carotid artery (p 0.03). However, in multivariate logistic regression models, none of the evaluated variables were significantly associated with IMT > 1. Conclusions: Our real-life data show that patients treated with PIs have a trend of developing both greater dyslipidemia and increased pathological IMT and atheromatous plaques These findings may be useful to optimize antiretrovirals for patients with cardiovascular risk factors.Background: Antiretroviral therapy has increasingly improved management of HIV infection, ensuring long-term efficacy and tolerability. Each class of antiretrovirals has, however, different characteristics and different tolerability profiles. The literature data show that protease inhibitors (PIs) are associated with a higher incidence of dyslipidemia. The aim of our study was to evaluate whether patients treated with PIs have both greater dyslipidemia and increased intima media thickness (IMT) and atheromatous plaques compared to patients treated without PIs. Materials and Methods: A total of 110 HIV-experienced patients screened with Doppler ultrasonography of the supra-aortic trunks in December 2019 were enrolled in a retrospective cross-sectional observational study. Patients were divided into two groups: 59 in the PI-based group, treated with PIs, and 51 in the PI-sparing group. In the two groups, we evaluated lipids, cardiovascular risk factors , increased pathological IMT , and possible atheromatous plaque. Results: Serum LDL ( The advent of protease inhibitors (PIs) has strongly changed the prognosis of patients with human immunodeficiency virus (HIV) infection. In fact, their high genetic barrier obtains long-term efficacy in the control of viral replication. However, treatment with PIs may induce dyslipidemia, which may be a problem for patients who become aged, who may have weight gain, and who may smoke and may develop atheromatous plaques , and thuCVD is one of the most important causes of mortality and morbidity in the industrialized world. People with hyper-lipidemia, in fact, are at roughly twice the risk of developing CVD compared to those with normal total cholesterol levels . In partIn the present study, we evaluated PLWHIV, treated in real life with different types of regimens and different PIs, used in triple or dual therapy, compared to PI-sparing regimens, including non-nucleoside reverse-transcriptase inhibitors (NNRTIs) and integrase inhibitors (INIs), and screened in December 2019 by Doppler ultrasonography of the supra-aortic trunks. The aim of our study was to analyze the possible association between the use of PIs in antiretroviral treatment, in any regimen, and the associated damage at the endothelium of supra-aortic trunks, expressed as pathological IMT and/or atheromatous plaques. We performed an observational, retrospective, cross-sectional study enrolling 110 HIV-experienced patients who were consecutively screened with Doppler ultrasonography of the supra-aortic trunks in December 2019 and treated with the same antiretroviral regimen. Patients were divided into 2 groups: 59 were treated with a PI-based antiretroviral regimen (PI-based group) and 51 were treated with an antiretroviral regimen without PIs (PI-sparing group). IMT of common and internal carotid for both left and right sides: ultrasonography of the epi-aortic vessels using a power color-Doppler instrument with 7.5 MHz probes. We evaluated the characteristics of the intima, together with the pulsation index, resistance index, minimal speed, peak speed and mean speed. A minimum of three measurements were required: on the common carotid artery, 1 cm before the carotid bifurcation and at the carotid bifurcation; on the internal carotid, 1 cm after the carotid bifurcation and 2 cm after the carotid bifurcation. The definition of IMT is the distance between the leading edge of the media–adventitia echo and the leading edge of the internal lumen. The larger IMT of the bilateral common carotid artery is the final value of the IMT. An IMT greater than 1 mm and/or atherosclerotic plaques are considered pathologic findings . AtherosData regarding independent risk factors for cardiovascular disease (CVD), such as family history, smoking, and diabetes.HIV viral load, CD4+ cell counts, total serum cholesterol, low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, glycemia, triglycerides, and body mass index (BMI) were recorded at each control visit.In the 2 groups of subjects, we evaluated the following parameters:All procedures performed in this study were in accordance with the 1964 Helsinki declaration and its later amendments or comparable ethics standards. Informed consent was obtained from all participants included in the study.The primary outcome was the evaluation of the development of pathologic IMT and its correlation with the PI-based regimen in HIV-experienced patients. The secondary outcome was the evaluation of the association between dyslipidemia and PI-based treatment. Dyslipidemia referred to unhealthy levels of one or more kinds of lipids in the blood  and/or use of statins. We also evaluated cardiovascular risk index (CRI), which was calculated by dividing total cholesterol level by HDL level: an increased CRI was considered for values > 5 in men and >4.5 in women . The HIV viral load was assessed by real-time PCR with the lowest detection limit of 40 copies/mL.Lymphocyte subsets  were evaluated with flow cytofluorimetry using monoclonal antibodies and a fluorescence-activated cell sorter scan . Serum lipids and routine analyses were performed applying standard procedures. t-test for comparison between two groups; in the case of non-normally distributed variables, the Mann–Whitney U test was performed; categorical variables were compared using the Chi-square or Fisher’s exact test. Continuous variables were summarized as mean and standard deviation or median at 25° and 75° quartile, and categorical variables as absolute and relative frequencies. To evaluate distribution of continuous variable we used the Shapiro–Wilk test, and to assess homoscedasticity we used Levene’s Test. For continuous variables, if normally distributed, the differences were evaluated by the Student’s p-value below 0.05 was considered statistically significant. Analyses were performed using STATA (version STATAC 16.1).We performed univariate and multivariate logistic regression models to investigate possible association between IMT > 1 and different assessed parameters. Multivariate regression models were adjusted for age, BMI, diabetes, ARV history, and PI-based regimen. Results of the logistic regressions are presented as odds ratio (OR) and 95% confidence interval (CI). A The characteristics at enrollment of the 110 PLWHIV included in the study are summarized in Of the 110 HIV-experienced patients enrolled in the study, 59 were included in the PI-based group, and 51 in the PI-sparing group. The subjects in the PI-based group were treated with PI for a median of 96 months, a mean of 103 , while those in the PI-sparing group were treated on antiretroviral therapy for a median of 96 months, a mean of 111.6 (±61.44 SD). Specifically, as regards the type of drugs used, in the PI-sparing group 13 patients were treated with integrase inhibitors (INIs) ; 38 were treated with NNRTIs . In the PI-based group, 46 patients were treated with a triple-therapy regimen containing PI . In the same group, 13 patients were treated with PI in dual therapy , while 1 (7.6%) was treated with a different dual regimen associating NNRTI (Etravirine) and DRV/r. The backbone of ART was similar between the 51 subjects in the PI-based group and the 46 in the PI-based group in triple ART therapy.p 0.002) (p 0.003)  . In the p 0.003) , but unip 0.003) .p 0.04), and the percentage of patients with hypercholesterolemia (p 0.03) was higher in the PI group  were similar in the two groups, except for median serum LDL (PI group . As regap 0.03)  (p 0.03) . The demp 0.03) .The advent of PIs has given HIV patients the hope of surviving but at the risk of developing some comorbidities related to drugs and/or to aging. Therefore, this prognostic improvement has forced clinicians to manage different comorbidities and above all long-term metabolic status. PIs in particular appear related to higher risk of dyslipidemia, which is a cardiovascular risk factor above all when it is associated with the development of atherosclerotic plaques in the supra-aortic trunks . We sought to demonstrate that there may be an association between PIs and cardiovascular risk with the evidence of dyslipidemia associated with a higher rate of pathological IMT. This alteration is preparatory to the development of atherosclerosis and CVD. Comparing Doppler data of patients treated with PIs vs. those without PIs, we highlighted that there is a correlation between these drugs and elevated IMT. This was statistically significant in particular for the section of the left internal carotid artery (ICA). The data in the literature on the association between PIs and CVD are conflicting. In 2005, Iloeie et al., evaluating 7542 patients, observed that patients exposed to PI therapy had an increased risk of CVD events, and therefore clinicians should evaluate the risk of CVD when making treatment decisions for HIV-infected patients . In reveAs regards the pathogenetic mechanisms of this association, some authors have hypothesized that the ART activates endothelial function and promotes atherosclerosis. It is possible that HIV, immune reconstitution response, and ART may promote early endothelial activation, and hence represent pro-atherogenic factors and/or accelerators of atherosclerosis ,20. AnalThe increased risk of atherosclerotic cardiovascular disease among HIV-infected patients receiving a PI-based regimen may not be directly due to the acceleration of the pro-inflammatory response. The major mechanism in which PI-based regimens were triggering atherogenesis could be through an alteration of lipid metabolism and endothelial function. In a recent review in 2021, Vos et al. concluded that PIs and efavirenz have associated metabolic disturbances and increased risk of CVD, although their use was decreasing worldwide . In 2014The authors hypothesized that PIs trigger reactive oxygen species (ROS) production, which leads to serious downstream consequences such as cell death, impaired mitochondrial function, and UPS dysregulation. HIV PI-induced ROS, together with a dysfunctional UPS, elicit definitive effects on the cardiovascular system that will eventually result in the onset of heart diseases. Thus, while PIs substantially improve life expectancy and quality of life in HIV-positive patients, their longer-term side-effects on the cardiovascular system should lead to (a) greater clinical awareness regarding the benefit–harm paradigm, and (b) the development and evaluation of novel co-treatment strategies. Analyzing data from the literature, it appears that recent papers, in contrast to the older ones, seem to give more importance to the role of HIV infection and PI-based regimens in the onset of higher IMT. These data may suggest that CVD is a risk factor related to the class of PI and to specific previous drugs. In fact, in a recent review of 2021, Hateleberg et al. concluded that although PIs will likely remain in use globally for several years to come, baseline CVD risk should be considered when selecting their use, especially as the population with HIV ages . This is the reason why more metabolic-friendly treatment regimens have been developed in recent years. The advent of INIs has allowed the adoption of two-drug regimens, with similar efficacy and a better tolerability profile than triple regimens ,27,28,29This study has some limitations, being a retrospective cross-sectional monocenter design, and moreover, the study was concluded in December 2019 and consequently no patient had been treated with bictegravir because this drug was not available at that time. There were 14 patients in the PI-sparing group with a previous history of PI-based therapy, but they represented only 27% of the group and the average duration of therapy with PI was only 18 months, probably insufficient to cause tangible endothelial damage.Furthermore, in the PI group, the authors used four different PI/r and an unboosted PI in five cases. There may be a potentially different cardiovascular risk associated with the different PIs adopted. For example, ATV was known to be a PI with lower metabolic impact and lower cardiovascular risk than other PIs , especiaIn conclusion, observing PLWHIV treated in real life, we noted more pathological IMT in patients treated with PI vs. PI-sparing regimens. This may be related to a higher risk of CVD in these patients, as shown in the literature. It is possible that endothelial damage is progressive, and in our study, patients in the PI group had been treated for longer than the PI-sparing group. In any case, this may further support the possibility of a proactive switch from PI to other drugs with a more metabolic-friendly profile for patients with a potential high risk of CVD, such as diabetics and the elderly.


Despite tremendous efforts, late presenters (CD4 ≤ 350 cells/μl) still account for a staggering proportion of HIV patients at the time of diagnosis. The main objective of this study was to evaluate the viral-immunological efficacy of bictegravir/emtricitabine/ tenofovir alafenamide (BIC/FTC/TAF) in treatment-naïve, late presenters in Chinese patients with HIV-1 infection, comparing with national free tenofovir disoproxil fumarate/lamivudine/efavirenz/(TDF/3TC/EFV).We conducted an open-label, randomized controlled trial at six HIV care centers in China, starting recruitment from June 2021 to December 2022. All treatment-naïve late presenters were randomised to receive TDF/3TC/EFV (group A) or BIC/FTC/TAF (group B). Serum HIV loads and CD4 cell counts were measured at baseline, 4 weeks, 12 weeks, and 24 weeks. The proportion of HIV-RNA < 50 copies/ml at 12 weeks was considered the primary endpoint and changes in viral loads and CD4 cell counts at other time points were secondary outcomes in PP and ITT analysis.We enrolled 200 late presenters, randomizing to group A (n=99) and group B (n=101). As of April 2023, 178/200 (89%) and 156/200 (78%) of randomized individuals completed 12 weeks and 24 weeks of follow-up. The study groups (n=200) were similar at baseline with a mean age of 43.0±13.8 years, 83.5% men, HBV co-infection percentage of 4%, HCV co-infection percentage of 2.5%, HIV viral load of mean 4.7±0.7 log copies/ml and CD4 cell counts of mean 109±74 cells/μl (Table 1). PP analysis showed 67.9% (57/84) of patients in group B had achieved viral suppression (< 50 copies/mL) at 12 weeks in comparison to 48.1 % (37/77) of that in group A (p< 0.01). The mean viral load levels decreased sharply in the drug B group from 4.70 ± 0.72 to 1.62 ± 0.47 log copies/ml over 12 weeks, compared to group A . However, in the two groups, CD4 counts had a similar change trend over 24 weeks and the proportion of CD4 cell counts over 200 cells/μl remained identical (figure 2). ITT analysis further demonstrated the results above.Compared to TDF/3TC/EFV, BIC/FTC/ TAF as the initial choice for late presenters had better performance to achieve rapid viral suppression, especially in the first three months therapy, but showed no superiority in immune deficiency improvement.All Authors: No reported disclosures


Background: Clinical trials and real-life studies have granted the efficacy and safety of dolutegravir and lamivudine (DTG/3TC) in naïve and experienced people living with HIV (PLWH), but there are no long-term data in elderly people. We herein describe our real-life cohort of PLWH who were ≥65 years of age (PLWH ≥ 65) who started or were switched to DTG/3TC, single-tablet regimen, or DTG plus 3TC. Methods: We considered laboratory/clinical parameter changes from the baseline to the last follow-up time point available for each person by the paired Wilcoxon test and analyzed factors associated with virological failure (VF) and discontinuation. Results: We included 112 PLWH with a median age of 66 (IQR: 65–70) years, 77.6% males; 84.8% of people had multimorbidity, 34.8% were on polypharmacy, and only 5.4% were naïve to treatment. Reasons to be switched to DTG/3TC were: abacavir removal (38.7%), treatment simplification (33.1%), and PI discontinuation (28.2%). The median treatment durability was 6 (IQR: 5.4–7) years. No significant changes were detected in metabolic, renal, immunological, or cardiovascular biomarkers during follow-up. HIV RNA undetectability was maintained in 104 (92.8%) individuals for whom follow-up evaluation was available. We observed eight discontinuations . No factors were significantly associated with VF or discontinuation. Conclusions: This is the first study on DTG/3TC in PLWH ≥ 65 with a follow-up longer than 5 years. DTG/3TC was found to be safe and effective, neutral on metabolic parameters, and with a low discontinuation rate for toxicity or VF. The pivotal moment in the evolution of antiretroviral therapy came in the mid-1990s with the advent of highly active antiretroviral therapy (HAART), a combination of multiple antiretroviral drugs from different classes to target various stages of the HIV lifecycle simultaneously . HAART mConsequently, in recent decades we have been witnessing the progressive aging of people living with HIV (PLWH) ,4. FurthRandomized and controlled clinical trials, as mentioned, but also real-life data have in recent years confirmed and sealed the efficacy of the dolutegravir (DTG) and lamivudine (3TC) regimen both in therapy switching, but also in naïve PLWH ,18. As t® dataset. We included demographics , HIV-related characteristics , laboratory findings , and anthropometric parameters . We categorized antiretroviral regimens at the baseline as: naïve ; mono/dual regimen ; 2 nucleos(t)ide reverse transcriptase inhibitors plus a protease inhibitor; 2 nucleos(t)ide reverse transcriptase inhibitors plus a non-nucleos(t)ide reverse transcriptase inhibitor; and 2 nucleos(t)ide reverse transcriptase inhibitors plus an integrase inhibitor. Reasons for switching to DTG/3TC were classified as: abacavir discontinuation (to reduce cardiovascular risk), protease inhibitor discontinuation (occurred due to dyslipidemia or drug–drug interactions), and simplification .This is a single-center, retrospective, observational study conducted at the Infectious and Tropical Diseases Unit of Padua University Hospital . It complies with principles of good clinical practice and was conducted in accordance with Declaration of Helsinki. The study protocol was provided to the Ethical Committee (study protocol n. 5285/AO). Participants’ consent was waived as per Italian law . We incl2) [We also included a list of the following comorbidities: malignancy, chronic renal disease (defined as an estimated glomerular filtration rate of <60 mL/min) , dyslipi2) , cirrhos2) ,23,24,25p-values < 0.1, the value set-up for statistical significance. The analyses were run through Stata SE 16 . Data are expressed as medians (interquartile range) or percentages, as appropriate. We recorded changes of any parameters from the baseline to the last follow-up time point available for each person and analyzed factors associated with virological failure (VF) and discontinuation. Continuous and categorical variables were reported as median values (interquartile range) and absolute numbers (proportion), respectively. The intra-subject median changes from baseline to follow-up were analyzed by the paired Wilcoxon test after excluding subjects classified as discontinuations. Binary and multinomial logistic regression was considered for univariate 3, while 24.1% of PLWH had experienced at least one AIDS-defining condition according to their past medical history. Six PLWH (5.4%) started this combination as a first-line regimen, while the remaining ones were switched off from other antiretrovirals. For these people, the zenith of HIV RNA was 74.100 (IQR: 63.000–86.700) copies/mL, and the median CD4+ T cell count at HIV diagnosis time was 451 (IQR: 240–748) cell/mm3. As for people who switched, HIV was very well controlled; 100% of such persons had HIV RNA levels of <50 copies/mL, and the median CD4+ T cell count was 645 (466–872) cell/mm3. The median duration of HIV infection was 25 (20–29) years, and the median CD4+ T cell count at nadir was 233 cell/mm3. Almost one out of four (24.1%) individuals had experienced an AIDS event in their past medical history. The vast majority of PLWH in our cohort presented multimorbidity (84.8%), while polypharmacy was present in 34.8% of cases. Each participant experienced a median number of four comorbidities (IQR: 2–6). The most common comorbidities were hypertension (50.9%), dyslipidemia (48.2%), osteoporosis (26.8%), cancer (24.1%), and chronic kidney disease (24.2%). Almost one of four (23.2%) and one of five (21.4%) PLWH in our cohort were affected by mood and neurological disorders, respectively. The most common reason to be switched to DTG+3TC was the removal of abacavir from the previous regimen (38.7%) due to high cardiovascular risk, followed by simplification (33.1%) and PI discontinuation (28.2%) due to high cardiovascular risk and metabolic toxicities. In addition, 47% of PLWH started this combination as a two-pill regimen, and all but six were switched to a single-tablet regimen as soon as it was available.From 8 April 2015 to 31 May 2023 (date of the last follow-up time point), 112 PLWH started DTG and 3TC in our center. In The median durability of follow-up of the DTG+3TC regimen was 6.0 (5.4–7.0) years. Along this period, we observed eight discontinuations: two participants who were deceased at weeks 70 and 95, respectively, for reasons not related to the antiretroviral regimen; two viral failures which occurred in two experienced people with no resistance selection at weeks 74 and 77; two interruptions which occurred due to neuropsychiatric intolerances at weeks 1 and 2; one due to significant weight gain (12 kg) at week 66; and one due to a person choosing to be switched to long-acting treatment at week 90. Seven discontinuations occurred in experienced people, while one occurred in a naïve person. One of the two cases of neuropsychiatric toxicity occurred in a naïve person. No clinical or laboratory parameters were associated with discontinuation. The use of cART in geriatric PLWH, defined as people aged 65 years and older, presents unique challenges and considerations due to the intersection of HIV infection and the complexities of aging. As the aging population living with HIV grows, optimizing treatment approaches for this specific population becomes increasingly important. Several studies have shed light on the benefits, challenges, and evolving strategies related to antiretroviral use in geriatric patients ,23,24,26Our real-life observational study showed that the combination of DTG and 3TC was effective and safe in elderly PLWH in the long-term follow-up. To our knowledge, it is the first report that documented such results in PLWH who are ≥65 years of age during a median follow-up time of 6 years.With advances in antiretroviral therapy and more potent, convenient, and tolerable regimens, virological suppression is easily achieved in most PLWH, and dual therapies have indeed been a major evolution over the triple-antiretroviral ones. Data are emerging on the safety and efficacy of other modern dual therapies based on INIs, such as dolutegravir and rilpivirine (RPV) or dolutegravir and doravirine (DOR) as switch strategies in people who have already gained virological suppression ,31,32,33The SWORD-1 and SWORD-2 studies granted the efficacy, tolerability and safety, and noninferiority of DTG/RPV compared to the standard triple therapy in maintaining virological suppression in PLWH who switch their regimen . RegardiIn our cohort, two persons experienced virological failure, but no one developed treatment-emergent resistance mutations at the time of virological failure, as already reported by a recent metanalysis . This alBeyond maintaining virological control, the main focus today in PLWH is to achieve and preserve well-being and good quality of life. This is particularly crucial in the aging population, whose prevalence is constantly increasing and will be higher than 70% in 2030 . MoreoveBy aging, PLWH experience a longer period of life characterized by multimorbidity, with complex drug regimens that will continue to be a challenge for clinical management, due to possible side effects, toxicity, and drug interactions ,11,24. IMultimorbidity and polypharmacy are of crucial concern for aging PLWH, whether they are antiretroviral-naïve or antiretroviral-experienced. Multimorbidity may drive and influence the choice of antiretroviral regimen and lead clinicians to select regimens that are both free from cardiovascular/kidney toxicity and metabolic/bone-friendly. In our study, the most important reason for switching to DTG+3TC was the removal of abacavir from the previous regimen due to high cardiovascular risk; the removal of protease inhibitors was also present in almost 30% of cases. Furthermore, multiple comorbidities lead to polypharmacy and render HIV-positive elderly people vulnerable to medication-related problems, including adverse drug reactions and potential drug–drug interactions . During Two persons in our study discontinued the DTG/3TC regimen early because of neuropsychiatric toxicities, which began soon after the regimen initiation. This area is of growing concern, as it is nowadays known that the emergence of neuropsychiatric toxicities is associated with the use of INIs, raising important questions about their safety and clinical implications over time ,50,51. NOn this topic, it has been observed that in geriatric PLWH, dolutegravir maximal concentrations were increased by 25%, even if this increase was not correlated with modification of sleep function . HoweverThis study is somewhat limited by the low number of participants and by its retrospective design. With a small sample size, it becomes challenging to generalize the findings to a larger population. The characteristics of the patients  in the study might not be representative of the broader patient population, making it difficult to apply the results to other settings or patient groups . It is crucial to include in such kinds of studies a larger proportion of post-menopausal women. Lastly, we did not include a control group following other antiretroviral regimens, whether dual or triple ones. Finally, even if longitudinal studies including a larger number of PLWH are needed to confirm our results, in this retrospective observational study the dual regimen of DTG/3TC has demonstrated its effectiveness in a geriatric cohort of PLWH in a real-world setting. The high rate of virological response, the good safety profile, and the low rate of drug–drug interactions together with the reduction in the daily number of antiretroviral drugs taken by PLWH makes this regimen particularly favorable for individuals with comorbidities and polypharmacy. It is worth noting how the use of antiretroviral therapy in geriatric patients requires a nuanced approach that considers both HIV-related factors and age-related considerations. Therefore, tailoring treatment regimens to the specific needs of geriatric patients is crucial for promoting healthy aging and improving their overall quality of life.


Leishmania parasite load determined using kinetoplast DNA (kDNA)-based quantitative polymerase chain reaction (qPCR) in visceral leishmaniasis (VL) patients.  This study aimed to describe the kinetics of  Parasite load in blood was assessed by qPCR at five time points, up to 12 months post-diagnosis. Sixteen patients were followed up.P < 0.0001). One patient had an increased parasite load 3 months post-treatment and relapsed clinically at month six.  A significant reduction in the parasite load was observed after treatment ( We have described the use of kDNA-based qPCR in the post-treatment follow-up of VL cases. Leishmania. In the Indian subcontinent, Asia, and Africa, VL is caused by Leishmania donovani and is an anthroponotic disease Leishmania infantum, which is mainly transmitted through the bites of female phlebotomine sand flies of the species Lutzomyia longipalpisVisceral leishmaniasis (VL) is a chronic, neglected disease caused by intracellular protozoa belonging to the genus Relapse is a new VL episode that occurs after an initial cure, usually within 12 months of the initial treatment-Leishmania kinetoplast DNA (kDNA) levels in peripheral blood at the time of diagnosis and during follow-up to evaluate this method for monitoring treatment relapses.One of the main challenges in the treatment of VL is the monitoring and prediction of relapse. Quantitative polymerase chain reaction (qPCR) using peripheral blood samples is an important tool for monitoring treatment responseThe study was conducted in a high-complexity hospital located in the city of Petrolina, Pernambuco, Brazil . The patients were followed-up during the infectious disease service of the municipality.Leishmania positivity in dogsPetrolina is located in the São Francisco Valley, in the Northeast Region of Brazil. It is considered a hyperendemic region for VL, with a mean incidence rate of 4.4 human cases per 100,000 inhabitants between 2007 and 2017The study population consisted of individuals aged ≥ 15 years  who sought health care for a clinical and laboratory condition suspected of VL between July 2017 and July 2020. The cases were confirmed by parasite visualization in bone marrow aspirates. Patients were included in the analysis if they had their blood samples collected before treatment and had at least one follow-up visit after treatment. All patients were tested for human immunodeficiency virus (HIV). Leishmania kDNA using qPCR. In addition, bone marrow samples were collected at baseline for parasite quantification.During follow-up, patients were clinically evaluated at baseline (T0), one day after treatment completion (T1), 3 months (T2), 6 months (T3), and 12 months (T4). Blood samples were collected at each visit for the quantification of This study was approved by the Ethics Committee of the Universidade Federal do Vale do São Francisco (CAAE:68562617.3.0000.5196).After diagnosis, all patients started VL therapy following the recommendations of the Brazilian Ministry of Health. In Brazil, the drugs used for the treatment of VL are N-methyl glucamine antimoniate and amphotericin B. For the choice of medication, age group, pregnancy, and comorbidities are considered. N-methyl glucamine antimoniate was administered at a dose of 20 mg/Sb+5/kg/day, intravenously or intramuscularly, once a day, for 30 days. Liposomal amphotericin B (L-AmB) was intravenously administered at a dose of 4 mg/kg/day for 5 days. Patients with HIV-LV co-infection received secondary prophylaxis with L-AmB at a dose of 4 mg/kg every 15 days. Clinical response was defined as remission of fever, improvement in hematological values, and regression in spleen and/or liver size during therapy. Clinical relapse was defined as the recurrence of VL symptoms and signs within 12 months of treatment.Samples were collected in EDTA tubes  and DNA extraction was performed using the PureLink Genomic DNA Mini Kit  according to the manufacturer’s instructions. The DNA samples were eluted to a final volume of 100 μL. The overall quality of DNA in terms of purity was assessed using a NanoDrop OneC spectrophotometer . DNA samples were frozen at −20 °C until analysis.Leishmania spp. kDNA by qPCR using the primers LEISH-1 (5’-AACTTTTCTGGTCCTCCGG GTAG-3’) and LEISH-2 (5’-ACCCCCAGTTTCCCGCC-3’), as well as the TaqMan-MGB probe (FAM-5’-AAAAATGGGTGCAGAAAT-3’-nonfluorescent quencher-MGB), as described previouslyDNA samples were screened for the presence of L. infantum promastigotes was used as a positive control and also to estimate the parasite load in the patient samples. DNA-free water was used as a non-template control. All qPCR reactions were performed in duplicates. A standard curve prepared using nine serial dilutions  of genomic DNA extracted from cultured P < 0.05.Statistical analyses and graph preparation were performed using GraphPad Prism version 8 . The Kolmogorov-Smirnov test was used to investigate the normality of the data distribution. Statistical differences in median parasite load were determined using the Wilcoxon signed-rank test. Statistical significance was set at During the study period, 38 patients were diagnosed with VL. Four (10.5%) patients died before the end of treatment . Sixteen patients fulfilled the inclusion criteria and were followed-up before (T0) and after treatment (T1). Nine patients were lost to follow-up at T2 (3 months), one at T3 (6 months), and only one who presented with clinical symptoms at T3 returned at T4 (12 months). The baseline clinical and laboratory characteristics of the patients are presented in Leishmania kDNA was detected in all 16 patients with VL at the time of diagnosis (T0), with a median of 2.56 log parasites/mL (range 0.71-4.09). The parasite load in the bone marrow of eight patients with available samples was found to be approximately two times higher than that observed in blood samples at T0 (P = 0.007) (= 0.007) , corroboThirteen patients were treated with L-AmB (81.2%) and three with N-methyl glucamine antimoniate . P < 0.0001) (P = 0.25)  . Two pat = 0.25) .At T2 and T3, the median parasite load was 0.00 and 0.08 log parasites/mL, respectively. Only patient number 11 had an increased parasite load at T2, but had no noticeable clinical signs or symptoms at the time of the visit. At T3, the patient returned to the clinic presenting with asthenia, weight loss, hepatomegaly, and worsening hematological parameters. The patient was treated again with L-AmB. At T4, after the second treatment, the patient no longer had symptoms and there was a reduction in the parasite load . The cliLeishmania kDNA detection by qPCR in blood samples may be a potential target in the diagnosis and post-treatment follow-up of patients in areas endemic for VL.The present study demonstrates that Leishmania kDNA levels at T1. At T2 (3 months), six of seven patients (85.7%) had undetectable Leishmania kDNA levels. Previous studies have demonstrated the usefulness of qPCR in estimating the parasite load in blood samples of VL patients and also observed that despite the significant reduction in parasite load at the end of treatment, it remains detectable in most patients-,We observed a significant reduction in the parasite load immediately after the end of treatment. Despite this reduction, only two patients (12.5%) had undetectable Leishmania DNA levels. Mary et al. (2004)®, which remained at less than 1 parasite/mL. In addition, Aoun et al. (2009)Bossolasco et al. (2003)Leishmania kDNA levels immediately after the completion of treatment. Despite the methodological differences between the studies , the treatment regimen may partly explain the differences in clearance time between the studies, as treatment with L-AmB is administered for 5 days, and treatment with meglumine antimoniate can range between 20 and 40 days. In this study, two of three (66.6%) patients treated with meglumine antimoniate had undetectable L. donovani parasitemia within 30 days of treatment with L-AmB in individuals who relapsed, corroborating other studies that observed an increase above 10 parasites/mL in blood before clinical relapse,In our study, a 67-year-old patient showed a 2-log increase in parasite load 3 months after treatment, and relapsed clinically at month six. Advanced age has been associated with relapse in immunocompetent patients treated with L-AmBL. infantum using L-AmB. This study presents data on the detection of Leishmania kDNA using qPCR in a series of cases followed-up longitudinally after therapy. One case of clinical relapse presented an increased parasite load before symptom onset. Further studies with larger populations are needed to evaluate the use of this technique, as well as the definition of a cutoff point, in the prediction of relapse in VL patients.This study had some limitations, including the small sample size and significant loss to follow-up. However, few studies have evaluated the role of therapeutic monitoring by qPCR in the blood of individuals infected with