Therapeutic drug monitoring and virological response at week 48 in a cohort of HIV-1-infected patients switching to dolutegravir/rilpivirine dual maintenance therapy (ANRS-MIE-BIRIDER study).

AIMS: Dolutegravir (DTG) and rilpivirine (RPV) dual therapy is now recommended as a switch option in virologically suppressed HIV patients. Literature suggests that virological failure with dual therapy could possibly relate to subtherapeutic drug concentrations. In this study, we aimed at describing the DTG and RPV trough plasma concentrations (Cmin) and plasma HIV-1 RNA viral load (VL) during maintenance dual therapy. METHODS: We performed a retrospective analysis of DTG and RPV therapeutic drug monitoring in people living with HIV/AIDS (PLWHA) with dual therapy in 9 French centres. DTG and RPV trough plasma concentrations were estimated using a Bayesian approach to predict Cmin. The relationship between the pharmacokinetics of DTG and RPV and VL > 50 copies (cp)/mL was explored using joint nonlinear mixed models. The frequency of subtherapeutic threshold (DTG Cmin below 640 ng/mL and RPV Cmin below 50 ng/mL) were compared between PLWHA presenting VL > 50 cp/mL or not during the study. RESULTS: At baseline, 209 PLWHA were enrolled in the study. At week 48, 19 people living with HIV/AIDS (9.1%) discontinued their treatment and 15 PLWHA (7.1%) exhibited VL > 50 cp/mL. Six PLWHA out of 15 (40.0%) with VL > 50 cp/mL during the follow-up had at least 1 Cmin below the respective thresholds while only 26/194 patients (13.4%) without virological replication had at least 1 concentration below the threshold (P = .015). CONCLUSION: A majority of PLWHA receiving DTG/RPV maintenance dual therapy demonstrated VL < 50 cp/mL but virological replication was more frequent in people living with HIV/AIDS with subtherapeutic Cmin.

Letermovir for CMV Prophylaxis in Very High-Risk Pediatric Hematopoietic Stem Cell Transplantation Recipients for Inborn Errors of Immunity.

The burden of CMV infection and disease is important in pediatric hematopoietic stem cell transplantation (HSCT), notably in the subgroup of patients with inborn errors of immunity (IEIs). Letermovir (LMV) is now a standard of care for CMV prophylaxis in adult sero-positive (R+) recipients, but is not yet labeled for children. Published pediatric studies are still scarce. We report a monocentric real-life use of LMV in 36 HSCT pediatric recipients with IEIs considered at high-risk of CMV infection including 14 patients between 2 and 12 months of age. A homogenous dosage proportional to the body surface area was used. Pharmacokinetic (PK) was performed in 8 patients with a median of 6 years of age (range 0,6;15). The cumulative incidence of clinically significant CMV infections (CS-CMVi) and the overall survival of patients under LMV were compared to a very similar historical cohort under (val)aciclovir prophylaxis. LMV tolerance was good. As compared to the historical cohort, the incidence of CS-CMVi was significantly lower in LMV group (5 out of 36 transplants (13.9%) versus 28 of the 62 HSCT (45.2%)) (p = 0.002). Plasma LMV exposures did not significantly differ with those reported in adult patients. In this high-risk pediatric HSCT cohort transplanted for IEIs, CMV prophylaxis with LMV at a homogenous dosage was well tolerated and effective in preventing CS-CMVi compared with a historical cohort.

Pharmaco-virological algorithm to target risk of drug resistance among a population of HIV-infected key populations in Togo.

No data about antiretroviral (ARV) treatment coverage and virological response are available among key populations (female sex workers [FSW] and Men having Sex with Men [MSM]) in Togo. This study aimed to both describe Human Immunodeficiency Virus (HIV) immunovirological status and evaluate the pertinence of an original algorithm combining pharmacology (PK) and viral load (VL) to identify subjects at risk of ARV drug resistance. A cross-sectional multicentric study was conducted in 2017 in Togo. Our PK-virological algorithm (PK-VA) defines subjects at risk of resistance when exhibiting both detectable plasma drug concentrations and VL > 200 c/mL. Among the 123 FSW and 136 MSM included, 50% and 66% were receiving ARV, with 69% and 80% of them successfully-treated, respectively. Genotypes showed drug-resistance mutation in 58% and 63% of nonvirologically controlled (VL > 200 c/mL) ARV-treated FSW and MSM, respectively. PK-VA would have enabled to save 75% and 72% of genotypic tests, for FSW and MSM, respectively. We reported first data about HIV care cascade among key populations in Togo, highlighting they are tested for HIV but linkage to care remains a concern. Furthermore, 70%-80% of ARV-treated participants experienced virological success. In limited resources settings, where genotyping tests are beyond reach, PK-VA might be an easiest solution to sort out patients needing ARV adaptation due to resistance.

Doravirine Exposure Decreased by Dialysis in a HIV Patient: A Grand Round.

BACKGROUND: The authors report the case of a 66-year-old male patient who was hemodialyzed 3 times per week for chronic renal failure and treated with 100 mg of doravirine once daily in combination with dolutegravir for HIV-1. No dose adjustment is required for doravirine in cases of severe renal injury, but the effect of dialysis on its exposure is poorly understood. METHODS RESULTS: Two series of 2 samples were drawn before and after 4-hour hemodialysis and showed an average doravirine concentration decrease of 48.1 ± 6.7%. The effects of hemodialysis were important, contrary to what was expected and has been previously reported. In addition, intraindividual variability was low. Nevertheless, because the concentrations reported were largely above the inhibitory concentration 50 (IC 50 ), no dose adjustment was required. CONCLUSIONS: The decrease in doravirine concentration due to hemodialysis observed in this case report was quite significant. Therefore, therapeutic drug monitoring might be recommended in certain patients undergoing doravirine treatment also on hemodialysis.

HIV-1 RNA Kinetics in Blood Plasma and in Seminal Plasma of Men Starting a Dolutegravir-Based Triple-Combination Regimen at the Time of Primary HIV-1 Infection.

We compared the proportion of participants achieving first undetectable HIV-1 RNA (VL) in seminal plasma (SP) and blood plasma (BP) in 19 men starting dolutegravir-based regimen at primary HIV infection. At baseline, median VL was 6.5 (interquartile range [IQR], 5.6-7.9) and 4.5 (IQR, 3.5-5.0) log10 copies/mL in BP and SP, respectively. Between baseline and week 48, significantly higher proportion of participants achieved first VL below limit of quantification in SP (93.0%) than in BP (84.2%; P = .008). Time to first undetectable VL was 8 weeks in SP (95% confidence interval [CI], 5.6-10.4) and 24 weeks in BP (95% CI, 14.1-33.9).

Impact of Loading Dose of Caspofungin in Pharmacokinetic-Pharmacodynamic Target Attainment for Severe Candidiasis Infections in Patients in Intensive Care Units: the CASPOLOAD Study.

This study evaluated the impact of a high loading dose of caspofungin (CAS) on the pharmacokinetics of CAS and the pharmacokinetic-pharmacodynamic (PK-PD) target attainment in patients in intensive care units (ICU). ICU patients requiring CAS treatment were prospectively included to receive a 140-mg loading dose of CAS. Plasma CAS concentrations (0, 2, 3, 5, 7, and 24 h postinfusion) were determined to develop a two-compartmental population PK model. A Monte Carlo simulation was performed and the probabilities of target attainment (PTAs) were computed using previously published MICs. PK-PD targets were ratios of area under the concentration-time curve from 0 to 24 h (AUC0-24h) divided by the MIC (AUC0-24h/MIC) of 250, 450, and 865 and maximal concentration (Cmax) divided by the MIC (Cmax/MIC) of 5, 10, 15, and 20. Among 13 included patients, CAS clearance was 0.98 ± 0.13 liters/h and distribution volumes were V1 = 9.0 ± 1.2 liters and V2 = 11.9 ± 2.9 liters. Observed and simulated CAS AUC0-24h were 79.1 (IQR 55.2; 108.4) and 81.3 (IQR 63.8; 102.3) mg · h/liter during the first 24 h of therapy, which is comparable to values usually observed in ICU patients at day 3 or later. PTAs were >90% for MICs of 0.19 and 0.5 mg/liter, considering AUC/MIC = 250 and Cmax/MIC = 10 as PK-PD targets, respectively. Thus, a high loading dose of CAS (140 mg) increased CAS exposure in the first 24 h of therapy, allowing early achievement of PK-PD targets for most Candida strains. Such a strategy seems to improve treatment efficacy, though further studies are needed to assess the impact on clinical outcomes. (This study has been registered at ClinicalTrials.gov under identifier NCT02413892.).

M184V/I does not impact the efficacy of abacavir/lamivudine/dolutegravir use as switch therapy in virologically suppressed patients.

BACKGROUND: M184V/I NRTI resistance mutations can be selected by either lamivudine/emtricitabine or abacavir. There are controversies about the use of abacavir/lamivudine/dolutegravir combinations in HIV-1-infected treatment-experienced patients with a fully suppressed HIV viral load (VL) and harbouring M184V/I. OBJECTIVES: We assessed the efficacy of abacavir/lamivudine/dolutegravir when used in HIV-infected pretreated patients with an undetectable VL who previously harboured M184V/I as a unique NRTI resistance mutation in a genotypic resistance test and had no resistance to integrase inhibitors. PATIENTS AND METHODS: A total of 154 patients with a fully suppressed HIV-1 plasma VL (<50 copies/mL) treated with tenofovir disoproxil fumarate/emtricitabine/boosted PI or abacavir/lamivudine/boosted PI who switched to an abacavir/lamivudine/dolutegravir regimen and had M184V/I as a unique NRTI resistance mutation in their therapeutic history were retrospectively analysed up to 12 months after the switch to abacavir/lamivudine/dolutegravir. Assessment of residual viraemia was performed at Months 1, 3, 6 and 12. Plasma VL with undetectable HIV-1 RNA corresponded to an absence of residual viraemia. RESULTS: During the 12 months of follow-up, three patients had a blip of VL (53, 62 and 106 copies/mL) at Month 3 followed by a subsequent VL <50 copies/mL. No patient harboured a virological failure during the follow-up. Moreover, there was no change in residual viraemia during the follow-up. CONCLUSIONS: M184V/I as a unique NRTI resistance mutation, regardless of possible selection by regimens containing lamivudine/emtricitabine or abacavir, does not affect the virological response of well-controlled patients who switched to abacavir/lamivudine/dolutegravir for at least 12 months.

Pharmacovirological analyses of blood and male genital compartment in patients receiving dolutegravir + lamivudine dual therapy as a switch strategy (ANRS 167 LAMIDOL trial).

OBJECTIVES: To describe plasma residual HIV viraemia, cellular HIV reservoir size, blood plasma drug concentrations and their male genital tract penetration during the maintenance dual therapy dolutegravir + lamivudine. PATIENTS AND METHODS: ANRS167 LAMIDOL enrolled 104 virologically suppressed patients to switch to dolutegravir + lamivudine. In this pharmacovirological substudy, ultrasensitive plasma viral load (USpVL) and plasma drug concentrations were measured at Day 0 (D0), Week 24 (W24) and W48 of dolutegravir + lamivudine, and HIV-DNA was measured at W-8 and W48. Semen samples were collected at D0 and W24 from 18 participants. Total and unbound blood and seminal plasma drug concentrations were measured using UPLC-MS/MS. RESULTS: Median HIV-DNA was 2.5 log10 copies/106 PBMC (IQR = 2.2-3.0, n = 100) at W-8 and 2.4 log10 copies/106 PBMC (IQR = 2.1-2.9, n = 100) at W48 (P = 0.17). The proportion of patients with undetected USpVL was 38% (n = 98), 43% (n = 98) and 49% (n = 97) at D0, W24 and W48, respectively (P = 0.08). Total and unbound plasma dolutegravir concentrations were stable between timepoints (P = 0.13) and all total plasma dolutegravir concentrations except one were adequate. Median free fraction of dolutegravir in plasma was 0.21%. Median blood plasma and seminal plasma concentrations of total dolutegravir at 24 h were 1812 ng/mL and 206 ng/mL, respectively. Median seminal plasma/blood plasma total concentration ratios were 11.6% and 2478% for dolutegravir and lamivudine, respectively. HIV-RNA (365 to 475 copies/mL) was detected in seminal plasma of one patient at D0 (5.9%) and of two patients at W24 (11.8%). CONCLUSIONS: These findings add further important information regarding the effectiveness of dolutegravir + lamivudine maintenance dual therapy in terms of plasma residual viraemia, cellular reservoir size and drug penetration in the male genital tract.

High virological suppression regardless of the genotypic susceptibility score after switching to a dolutegravir-based regimen: week 48 results in an observational cohort.

Objectives: To assess, in a clinical cohort, the efficacy of switching current ART in virologically suppressed patients to a dolutegravir-based regimen, regardless of the genotypic susceptibility score (GSS). Patients and methods: This was an observational single-centre study assessing ART-treated patients with plasma viral load (pVL) <50 copies/mL who were switched to a dolutegravir-based regimen with 1 year of follow-up. PCR negative was defined as an undetected PCR signal. Trough plasma concentration (C24) was determined using UPLC-MS/MS. Results: Two hundred and thirty-nine patients initiated a dolutegravir-based regimen: 12%, 29% and 59% had a total GSS of 1 or 1.5 (group 1), 2 or 2.5 (group 2) and 3 (group 3), respectively. At switch initiation, the median time since first ART and the median duration with pVL <50 copies/mL were 13 years (IQR = 6-19) and 3 years (IQR = 1-6), respectively. Median times since last genotype were 9, 10 and 5 years for groups 1, 2 and 3, respectively. Twenty patients (8.4%) discontinued the dolutegravir-based regimen due to adverse events. During the study, 96.4% (n = 661/686) of all pVL were <50 copies/mL. Four patients (1.7%) experienced virological failure (two pVL >50 copies/mL) without emergence of resistance; these patients' GSSs were 2, 2.5, 3 and 3. The median dolutegravir C24 was 1545 ng/mL (IQR = 1150-2097). Of the patients with pVL <20 copies/mL, 72% were PCR negative during the follow-up, with no difference between the three groups of patients. Conclusions: This observational cohort study showed a high level of virological suppression maintenance in the first year following the switch to a dolutegravir-based regimen, even in patients with GSS ≤2.

Pharmacokinetic modelling of darunavir/ritonavir dose reduction (800/100 to 400/100 mg once daily) in a darunavir/ritonavir-containing regimen in virologically suppressed HIV-infected patients: ANRS 165 DARULIGHT sub-study.

Background: In the ANRS 165 DARULIGHT study (NCT02384967) carried out in HIV-infected patients, the use of a darunavir/ritonavir-containing regimen with a switch to a reduced dose of darunavir maintained virological efficacy (≤50 copies/mL) for 48 weeks with a good safety profile. Objectives: To assess the total and unbound blood plasma pharmacokinetics of darunavir and associated antiretrovirals, and their penetration into semen before and after dose reduction. Patients and methods: Patients receiving a darunavir/ritonavir (800/100 mg q24h)-containing regimen for >6 months with plasma HIV-RNA ≤50 copies/mL for >12 months were switched to 400/100 mg darunavir/ritonavir q24h at week 0. A 24 h intensive pharmacokinetic blood sampling and a trough seminal sampling were performed before (week 0) and after (week 12) dose reduction. Individual pharmacokinetic parameter estimates were obtained using non-linear mixed-effect modelling for darunavir/ritonavir in blood plasma and used to test for bioequivalence, whereas darunavir/ritonavir in seminal plasma and NRTIs were analysed using a non-compartmental approach. Results and conclusions: Fifteen patients completed the intensive pharmacokinetic analysis. There was no significant decrease in total and unbound darunavir blood plasma exposure despite a 50% decrease in darunavir daily dose from 800 to 400 mg (AUC0-24 = 65 563 versus 52 518 ng·h/mL; P = 0.25). A decrease in apparent oral clearance (CL/F) of both darunavir and ritonavir at week 12 suggests a modification of the initial darunavir/ritonavir daily dose balance (800/100 to 400/100 mg), in favour of a reduced inducer effect of darunavir on cytochrome P450 and efflux transporters compared with the standard dose.

Impact of obesity on antiretroviral pharmacokinetics and immuno-virological response in HIV-infected patients: a case-control study.

Background: Obesity has high prevalence among HIV-infected patients. Increased adipose tissue mass affects the pharmacokinetics of numerous drugs, but few data are available for antiretroviral drugs. Objectives: In this study we aimed to explore the pharmacokinetics of antiretroviral drugs and the immuno-virological response in obese patients with HIV infection. Patients and methods: We examined data from 2009 to 2012 in our hospital's database for HIV-1-infected patients who received an antiretroviral drug (abacavir, emtricitabine, lamivudine, tenofovir, efavirenz, etravirine, nevirapine, atazanavir/ritonavir, darunavir/ritonavir, lopinavir/ritonavir or raltegravir). Obese patients were defined as those with BMI ≥30 kg/m 2 and normal-weight patients as those with BMI 19-25 kg/m 2 . Plasma concentrations ( C 12/24 ) were compared for each antiretroviral drug using a Mann-Whitney test. Suboptimal dosing and virological outcome were assessed by logistic regression, adjusting on covariates. Results: We enrolled 291 obese and 196 normal-weight patients. Among the 12 analysed antiretroviral drugs, tenofovir, efavirenz and lopinavir C 12 values were significantly lower in obese than normal-weight patients: 66 versus 86 ng/mL, 1498 versus 2034 ng/mL and 4595 versus 6420 ng/mL, respectively ( P < 0.001). Antiretroviral drug C 12/24 values were more frequently below efficacy thresholds for obese than for normal-weight patients after adjustment for other covariates ( P < 0.001). Although obese patients showed a higher CD4 count than normal-weight patients (510 versus 444 cells/mm 3 , P < 0.001), the groups did not differ in virological failure rate. Conclusions: This study highlights the impact of obesity on antiretroviral drug plasma exposure, but identifies no consequence of this suboptimal exposure on the immuno-virological control in this population.

Penetration and antiviral efficacy of total and unbound maraviroc, raltegravir and rilpivirine in both female and male genital fluids from HIV-positive patients receiving regimens containing these antiretrovirals.

BACKGROUND: Sub-optimal penetration of antiretroviral drugs in genital compartments might promote local HIV persistence and increase the risk of HIV transmission. OBJECTIVES: To describe the penetration of maraviroc, raltegravir, raltegravir glucuronide and rilpivirine in seminal plasma and cervico-vaginal secretions (CVS) and to assess local antiretroviral efficacy in HIV-1-positive patients. METHODS: This was a prospective, multicentre study. Inclusion criteria were HIV-1 positive, age >18 years, receiving regimens containing maraviroc and/or raltegravir and/or rilpivirine for >1 month, and good self-reported adherence. Paired blood and genital samples were collected 12 h (raltegravir and maraviroc) or 24 h (rilpivirine) post-dose. These concentrations were determined (UPLC-MS/MS) in blood and seminal plasma (total and unbound) and CVS (total, dried spots) and HIV-RNA was quantified in paired blood and genital samples. RESULTS: Among the 54 enrolled patients, 15 received maraviroc (6 men), 27 received raltegravir (14 men) and 20 received rilpivirine (10 men), corresponding to 54 total and 52 unbound plasma concentrations, 29 total CVS samples and 23 total and 18 unbound seminal plasma samples. Maraviroc and raltegravir displayed a ratio of genital fluids/plasma concentrations >0.5 in both male and female genital tracts. Conversely, rilpivirine displayed a low ratio. Antiretroviral free fractions were consistent with historical data. Nine patients had blood plasma HIV-RNA >50 copies/mL (2/9 had sub-optimal antiretroviral blood plasma exposure) and two other patients had detectable HIV-RNA in genital fluids. CONCLUSIONS: Maraviroc and raltegravir demonstrated good penetration in genital compartments, yielding good local virological response in genital compartments, whereas rilpivirine presented a low penetration profile but good local response.

Viral suppression and HIV-1 drug resistance 1 year after pragmatic transitioning to dolutegravir first-line therapy in Malawi: a prospective cohort study.

BACKGROUND: Many countries are now replacing non-nucleoside reverse transcriptase inhibitor (NNRTI)-based first-line antiretroviral therapy (ART) with a regimen containing tenofovir disoproxil fumarate, lamivudine, and dolutegravir (TLD). Recognising laboratory limitations, Malawi opted to transition those on NNRTI-based first-line ART to TLD without viral load testing. We aimed to assess viral load and HIV drug resistance during 1 year following transition to TLD without previous viral load testing. METHODS: In this prospective cohort study, we monitored 1892 adults transitioning from NNRTI-based first-line ART to the TLD regimen in the Médecins Sans Frontières-supported decentralised HIV programme in Chiradzulu District, Malawi. Eligible adults were enrolled between Jan 17 and May 11, 2019, at Ndunde and Milepa health centres, and between March 8 and May 11, 2019, at the Boma clinic. Viral load at the start of the TLD regimen was assessed retrospectively and measured at month 3, 6, and 12, and additionally at month 18 for those ever viraemic (viral load ≥50 copies per mL). Dolutegravir minimal plasma concentrations (Cmin) were determined for individuals with viraemia. Drug-resistance testing was done at the start of TLD regimen and at viral failure (viral load ≥50 copies per mL, followed by viral load ≥500 copies per mL; resistance defined as Stanford score ≥15). FINDINGS: Of 1892 participants who transitioned to the TLD regimen, 101 (5·3%) were viraemic at TLD start. 89 of 101 had drug-resistance testing with 31 participants (34·8%) with Lys65Arg mutation, 48 (53·9%) with Met184Val/Ile, and 42 (40·4%) with lamivudine and tenofovir disoproxil fumerate dual resistance. At month 12 (in the per-protocol population), 1725 (97·9% [95% CI 97·1-98·5]) of 1762 had viral loads of less than 50 copies per mL, including 83 (88·3% [80·0-94·0]) of 94 of those who were viraemic at baseline. At month 18, 35 (97·2% [85·5-99·9]) of 36 who were viraemic at TLD start with lamivudine and tenofovir disoproxil fumarate resistance and 27 (81·8% [64·5-93·0]) of 33 of those viraemic at baseline without resistance had viral load suppression. 14 of 1838 with at least two viral load tests upon transitioning had viral failure (all with at least one dolutegravir Cmin value <640 ng/mL; active threshold), suggesting suboptimal adherence. High baseline viral load was associated with viral failure (adjusted odds ratio [aOR] 14·1 [2·3-87·4] for 1000 to <10 000 copies per mL; aOR 64·4 [19·3-215·4] for ≥10 000 copies per mL). Two people with viral failure had dolutegravir resistance at 6 months (Arg263Lys or Gly118Arg mutation), both were viraemic with lamivudine and tenofovir disoproxil fumarate resistance at baseline. INTERPRETATION: High viral load suppression 1 year after introduction of the TLD regimen supports the unconditional transition strategy in Malawi. However, high pre-transition viral load, ongoing adherence challenges, and possibly existing nucleoside reverse transcriptase inhibitor resistance can lead to rapid development of dolutegravir resistance in a few individuals. This finding highlights the importance of viral load monitoring and dolutegravir-resistance surveillance after mass transitioning to the TLD regimen. FUNDING: Médecins Sans Frontières. TRANSLATIONS: For the French and Portuguese translations of the abstract see Supplementary Materials section.

Placental transfer of the integrase strand inhibitors cabotegravir and bictegravir in the ex-vivo human cotyledon perfusion model.

: Data on placental transfer is lacking for the recent HIV integrase inhibitors, bictegravir and cabotegravir, although their future use in pregnancy is to be expected. The objective of this study was to determine their transplacental pharmacokinetics. Maternal-to-fetal transfer was investigated using the open-circuit ex-vivo dually perfused human cotyledon model. Cabotegravir or bictegravir was added to a maternal perfusate containing 2 g/l of human albumin and antipyrine, a marker to validate the cotyledon's viability, and cotyledons were dually perfused for up to 90 min. For cabotegravir, in five experiments, the median (IQR 25-75) concentrations in the maternal and in the fetal compartments were, respectively, 550 ng/ml (344-788) and 48 ng/ml (37-54), with a maternal-to-fetal ratio of 10% (5-16) and a clearance index (in comparison with antipyrine transfer) of 22% (19-28). The median cotyledon accumulation index was 10% (2-21). For bictegravir, in six experiments, the median (IQR 25-75) concentrations in the maternal and in the fetal compartments were, respectively, 1650 ng/ml (1455-1960) and 126 ng/ml (112-142), with a maternal-to-fetal ratio of 7% (6-9.5) and a clearance index (in comparison with antipyrine transfer) of 21% (17-29). The median cotyledon accumulation index was 4% (3-5). Placental transfer of cabotegravir and bictegravir were low. This may not only limit the potential for fetal toxicities but also be a limit to their usefulness at the time of labor and delivery to reduce the risk of vertical HIV transmission. The safety and efficacy of these new integrase inhibitors in pregnancy require more investigation.