Persistent and multiclonal malaria parasite dynamics despite extended artemether-lumefantrine treatment in children.

Standard diagnostics used in longitudinal antimalarial studies are unable to characterize the complexity of submicroscopic parasite dynamics, particularly in high transmission settings. We use molecular markers and amplicon sequencing to characterize post-treatment stage-specific malaria parasite dynamics during a 42 day randomized trial of 3- versus 5 day artemether-lumefantrine in 303 children with and without HIV (ClinicalTrials.gov number NCT03453840). The prevalence of parasite-derived 18S rRNA is >70% in children throughout follow-up, and the ring-stage marker SBP1 is detectable in over 15% of children on day 14 despite effective treatment. We find that the extended regimen significantly lowers the risk of recurrent ring-stage parasitemia compared to the standard 3 day regimen, and that higher day 7 lumefantrine concentrations decrease the probability of ring-stage parasites in the early post-treatment period. Longitudinal amplicon sequencing reveals remarkably dynamic patterns of multiclonal infections that include new and persistent clones in both the early post-treatment and later time periods. Our data indicate that post-treatment parasite dynamics are highly complex despite efficacious therapy, findings that will inform strategies to optimize regimens in the face of emerging partial artemisinin resistance in Africa.

High-parameter phenotypic characterization reveals a subset of human Th17 cells that preferentially produce IL-17 against M. tuberculosis antigen.

Background: Interleukin-17-producing CD4 T cells contribute to the control of Mycobacterium tuberculosis (Mtb) infection in humans; whether infection with human immunodeficiency virus (HIV) disproportionately affects distinct Th17-cell subsets that respond to Mtb is incompletely defined. Methods: We performed high-definition characterization of circulating Mtb-specific Th17 cells by spectral flow cytometry in people with latent TB and treated HIV (HIV-ART). We also measured kynurenine pathway activity by liquid chromatography-mass spectrometry (LC/MS) on plasma and tested the hypothesis that tryptophan catabolism influences Th17-cell frequencies in this context. Results: We identified two subsets of Th17 cells: subset 1 defined as CD4+Vα7.2-CD161+CD26+and subset 2 defined as CD4+Vα7.2-CCR6+CXCR3-cells of which subset 1 was significantly reduced in latent tuberculosis infection (LTBI) with HIV-ART, yet Mtb-responsive IL-17-producing CD4 T cells were preserved; we found that IL-17-producing CD4 T cells dominate the response to Mtb antigen but not cytomegalovirus (CMV) antigen or staphylococcal enterotoxin B (SEB), and tryptophan catabolism negatively correlates with both subset 1 and subset 2 Th17-cell frequencies. Conclusions: We found differential effects of ART-suppressed HIV on distinct subsets of Th17 cells, that IL-17-producing CD4 T cells dominate responses to Mtb but not CMV antigen or SEB, and that kynurenine pathway activity is associated with decreases of circulating Th17 cells that may contribute to tuberculosis immunity.

Determination of sulfadoxine and pyrimethamine in microvolume human plasma using ultra high performance liquid chromatography-tandam mass spectrometry.

To support the pharmacokinetic study of sulfadoxine (SD) and pyrimethamine (PM) in pregnant women and children, sensitive methods with small sample volume are desirable. Here we report a method to determine SD and PM with microvolume plasma samples: 5 µL plasma samples were cleaned up by protein precipitation with acetonitrile. The deuterated analytes were used as the internal standards. The samples after cleanup were injected onto an ACE Excel SuperC18 column (50 × 2.1 mm, 1.7 µm, Hichrom Limited) connected to a Waters I class UPLC coupled with a Sciex Triple Quad 6500+ Mass Spectrometer and eluted with water and acetonitrile both containing 0.1% formic acid in a gradient mode at 0.8mL/min. Detection utilized ESI+ as the ion source and MRM as the quantification mode. The precursor-to-product ion transitions m/z 311→245 for SD and 249→233 for PM were selected for quantification. The ion transitions for the corresponding internal standards were 315→249 for SD-d4 and 254→235 for PM-d3. The simplest linear regression weighted by 1/x was used for the calibration curves. The calibration ranges were 1-200 µg/mL SD and 2 - 1000ng/mL PM. The mean (± standard deviation) recoveries were 94.3±3.2% (SD) and 97.0±1.5% (PM). The validated method was applied to analysis of 1719 clinical samples, demonstrating the method is suitable for the pharmacokinetic study with samples collected up to day 28 post-dose.

High-parameter phenotypic characterization reveals a subset of human Th17 cells that preferentially produce IL17 against M. tuberculosis antigen.

Background: Interleukin 17 producing CD4 T cells contribute to the control of Mycobacterium tuberculosis (Mtb) infection in humans; whether infection with Human Immunodeficiency Virus (HIV) disproportionately affects distinct Th17 cell subsets that respond to Mtb is incompletely defined. Methods: We performed high-definition characterization of circulating Mtb-specific Th17 cells by spectral flow cytometry in people with latent TB and treated HIV (HIV-ART). We also measured kynurenine pathway activity by LC/MS on plasma and tested the hypothesis that tryptophan catabolism influences Th17 cell frequencies in this context. Results: We identified two subsets of Th17 cells: subset 1 defined as CD4+Vα7.2-CD161+CD26+ and subset 2 defined as CD4+Vα7.2-CCR6+CXCR3- cells of which subset 1 was significantly reduced in LTBI with HIV-ART, yet Mtb-responsive IL17-producing CD4 T cells were preserved; we found that IL17-producing CD4 T cells dominate the response to Mtb antigen but not CMV antigen or staphylococcal enterotoxin B (SEB); and tryptophan catabolism negatively correlates with both subset 1 and subset 2 Th17 cell frequencies. Conclusions: We found differential effects of ART-suppressed HIV on distinct subsets of Th17 cells, that IL17-producing CD4 T cells dominate responses to Mtb but not CMV antigen or SEB, and that kynurenine pathway activity is associated with decreases of circulating Th17 cells that may contribute to tuberculosis immunity.

Seasonal Malaria Chemoprevention Drug Levels and Drug Resistance Markers in Children With or Without Malaria in Burkina Faso: A Case-Control Study.

BACKGROUND: Despite scale-up of seasonal malaria chemoprevention (SMC) with sulfadoxine-pyrimethamine and amodiaquine (SP-AQ) in children 3-59 months of age in Burkina Faso, malaria incidence remains high, raising concerns regarding SMC effectiveness and selection of drug resistance. Using a case-control design, we determined associations between SMC drug levels, drug resistance markers, and presentation with malaria. METHODS: We enrolled 310 children presenting at health facilities in Bobo-Dioulasso. Cases were SMC-eligible children 6-59 months of age diagnosed with malaria. Two controls were enrolled per case: SMC-eligible children without malaria; and older (5-10 years old), SMC-ineligible children with malaria. We measured SP-AQ drug levels among SMC-eligible children and SP-AQ resistance markers among parasitemic children. Conditional logistic regression was used to compute odds ratios (ORs) comparing drug levels between cases and controls. RESULTS: Compared to SMC-eligible controls, children with malaria were less likely to have any detectable SP or AQ (OR, 0.33 [95% confidence interval, .16-.67]; P = .002) and have lower drug levels (P < .05). Prevalences of mutations mediating high-level SP resistance were rare (0%-1%) and similar between cases and SMC-ineligible controls (P > .05). CONCLUSIONS: Incident malaria among SMC-eligible children was likely due to suboptimal levels of SP-AQ, resulting from missed cycles rather than increased antimalarial resistance to SP-AQ.

Efavirenz-Based Antiretroviral Therapy but Not Pregnancy Increased Unbound Piperaquine Exposure in Women during Malaria Chemoprevention.

Dihydroartemisinin-piperaquine (DP) is highly effective for malaria chemoprevention during pregnancy, but the standard dosing of DP that is used for nonpregnant adults may not be optimal for pregnant women. We previously reported that the pharmacokinetic exposure of total piperaquine (PQ; both bound and unbound to plasma proteins) is reduced significantly in the context of pregnancy or efavirenz (EFV)-based antiretroviral therapy (ART). However, as PQ is >99% protein-bound, reduced protein binding during pregnancy may lead to an increase in the pharmacologically active unbound drug fraction (fu), relative to the total PQ. We investigated the impact of pregnancy and EFV use on the fu of PQ to inform the interpretation of pharmacokinetics. Plasma samples from 0 to 24 h after the third (final) DP dose were collected from pregnant women at 28 weeks gestation who were receiving or not receiving EFV-based ART as well as from women 34 to 54 weeks postpartum who were not receiving EFV-based ART, who served as controls. Unbound PQ was quantified via ultrafiltration and liquid chromatography-tandem mass spectrometry, with fu being calculated as PQunbound/PQtotal. The geometric mean fu did not differ between pregnant and postpartum women (P = 0.66), but it was 23% (P < 0.01) greater in pregnant women receiving EFV-based ART, compared to that in postpartum women who were not receiving EFV-based ART. The altered drug-protein binding, potentially due to the displacement of PQ from plasma proteins by EFV, resulted in only a 14% lower unbound PQ exposure (P = 0.13) in the presence of a 31% lower total PQ exposure (P < 0.01), as estimated by the area under the concentration time curve from 0 to 24 h post-last dose in pregnant women who were receiving EFV-based ART. The results suggest that the impact of pregnancy and EFV-based ART on the exposure and, in turn, the efficacy of PQ for malaria prevention may not be as significant as was suggested by the changes in the total PQ exposure. Further study during the terminal elimination phase (e.g., on day 28 post-dose) would help better characterize the unbound PQ exposure during the full dosing interval and, thus, the overall efficacy of PQ for malaria chemoprevention in this special population.

Evaluating the effect of atorvastatin exposure and vitamin D levels on lipid outcomes in people with HIV-1 with suppressed HIV-1 RNA and LDL cholesterol <130 mg/dL.

INTRODUCTION: Cardiovascular disease (CVD) has become a leading cause of morbidity and mortality among people with HIV. Atorvastatin is known to reduce cardiovascular risk. We (1) compared atorvastatin concentrations between different boosted protease inhibitors (PIs) and with lipid outcomes and (2) compared pre-atorvastatin 25-OH vitamin D levels with atorvastatin concentrations and with lipid outcomes, in people with HIV with suppressed HIV-1 RNA and low-density lipoprotein cholesterol (LDL-C) <130 mg/dL. METHODS: A5275 was a randomized, double-blind, placebo-controlled crossover study of atorvastatin in virally suppressed people with HIV with fasting LDL-C <130 mg/dL. We analyzed results over the 20 weeks of active atorvastatin treatment. Atorvastatin was initiated at 10 mg daily and increased to 20 mg daily after 4 weeks if there were no findings of toxicity. Atorvastatin trough concentrations were measured at week 20. Participants took combination antiretroviral therapy (ART) that included a boosted PI throughout. RESULTS: Overall (n = 67), 70% of participants were male, and the median age was 51 years. There was no apparent association between atorvastatin trough concentrations and pre-atorvastatin vitamin D levels (r = 0.01, p = 0.9) or by boosted PI (p = 0.20). Median pre- to post-atorvastatin change was -39.0 mg/dL in fasting total cholesterol, -40.4 ng/mL in lipoprotein-associated phospholipase A2 (LP-PLA2), and -13.8 U/L in oxidized LDL, with all changes negatively correlated with atorvastatin trough concentrations (r = -0.19, -0.09, -0.21; p ≥ 0.096). CONCLUSIONS: No apparent associations between pre-atorvastatin vitamin D levels and outcomes were observed (all p > 0.70). In virologically suppressed people with HIV, higher atorvastatin concentrations were marginally associated with greater decreases in lipid outcomes.

Impact of Drug Exposure on Resistance Selection Following Artemether-Lumefantrine Treatment for Malaria in Children With and Without HIV in Uganda.

Artemisinin-based combination therapies (ACTs) are the primary treatment for malaria. It is essential to characterize the pharmacokinetics (PKs) and pharmacodynamics (PDs) of ACTs in vulnerable populations at risk of suboptimal dosing. We developed a population PK/PD model using data from our previous study of artemether-lumefantrine in HIV-uninfected and HIV-infected children living in a high-transmission region of Uganda. HIV-infected children were on efavirenz-, nevirapine-, or lopinavir-ritonavir-based antiretroviral regimens, with daily trimethoprim-sulfamethoxazole prophylaxis. We assessed selection for resistance in two key parasite transporters, pfcrt and pfmdr1, over 42-day follow-up and incorporated genotyping into a time-to-event model to ascertain how resistance genotype in relation to drug exposure impacts recurrence risk. Two hundred seventy-seven children contributed 364 episodes to the model (186 HIV-uninfected and 178 HIV-infected), with recurrent microscopy-detectable parasitemia detected in 176 episodes by day 42. The final model was a two-compartment model with first-order absorption and an estimated age effect on bioavailability. Systemic lumefantrine exposure was highest with lopinavir-ritonavir, lowest with efavirenz, and equivalent with nevirapine and HIV-uninfected children. HIV status and lumefantrine concentration were significant factors associated with recurrence risk. Significant selection was demonstrated for pfmdr1 N86 and pfcrt K76 in recurrent infections, with no evidence of selection for pfmdr1 Y184F. Less sensitive parasites were able to tolerate lumefantrine concentrations ~ 3.5-fold higher than more sensitive parasites. This is the first population PK model of lumefantrine in HIV-infected children and demonstrates selection for reduced lumefantrine susceptibility, a concern as we confront the threat to ACTs posed by emerging artemisinin resistance in Africa.

The Impact of Extended Treatment With Artemether-lumefantrine on Antimalarial Exposure and Reinfection Risks in Ugandan Children With Uncomplicated Malaria: A Randomized Controlled Trial.

BACKGROUND: Artemether-lumefantrine (AL) is the most widely used artemisinin-based combination therapy in Sub-Saharan Africa and is threatened by the emergence of artemisinin resistance. Dosing is suboptimal in young children. We hypothesized that extending AL duration will improve exposure and reduce reinfection risks. METHODS: We conducted a prospective, randomized, open-label pharmacokinetic/pharmacodynamic study of extended duration AL in children with malaria in high-transmission rural Uganda. Children received 3-day (standard 6-dose) or 5-day (10-dose) AL with sampling for artemether, dihydroartemisinin, and lumefantrine over 42-day clinical follow-up. Primary outcomes were (1) comparative pharmacokinetic parameters between regimens and (2) recurrent parasitemia analyzed as intention-to-treat. RESULTS: A total of 177 children aged 16 months to 16 years were randomized, contributing 227 total episodes. Terminal median lumefantrine concentrations were significantly increased in the 5-day versus 3-day regimen on days 7, 14, and 21 (P < .001). A predefined day 7 lumefantrine threshold of 280 ng/mL was strongly predictive of recurrence risk at 28 and 42 days (P < .001). Kaplan-Meier estimated 28-day (51% vs 40%) and 42-day risk (75% vs 68%) did not significantly differ between 3- and 5-day regimens. No significant toxicity was seen with the extended regimen. CONCLUSIONS: Extending the duration of AL was safe and significantly enhanced overall drug exposure in young children but did not lead to significant reductions in recurrent parasitemia risk in our high-transmission setting. However, day 7 levels were strongly predictive of recurrent parasitemia risk, and those in the lowest weight-band were at higher risk of underdosing with the standard 3-day regimen. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov number NCT03453840.

Quantification of N, N' N"-triethylenethiophosphoramide, N, N"-triethylenephosphoramide, cyclophosphamide, and 4-hydroxy-cyclophosphamide in microvolume human plasma to support neonatal and pediatric drug studies.

N, N' N"-triethylenethiophosphoramide (thiotepa) and cyclophosphamide (CP) are alkylating agents used for a variety of malignant and non-malignant disorders. Both drugs are metabolized by cytochrome P450 enzymes to form active metabolites. To support pharmacokinetic studies of thiotepa and CP in children, we sought to develop assays to determine parent drug and metabolite concentration in small volume plasma samples. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used for assay development. CP metabolite 4-hydroxycyclophosphamide (4OHCP) was converted to the more stable semicarbazone derivative (4OHCP-SCZ) for quantitation. Samples (10 µL) were extracted by solid-phase extraction and injected onto the LC-MS/MS system equipped with a pentafluorophenyl reverse phase column (2.1 × 50 mm, 2.7 µm). Electrospray ionization in positive mode was used for detection. Multiple reaction monitoring of the precursor-to-product ion transitions m/z 190→147 for thiotepa, 174→131 for tepa, 261→233 for CP, and 334→221 for 4OHCP-SCZ was selected for quantification. The ion transitions m/z 202→155 for thiotepa-d12, 186→139 for tepa-d12, 267→237 for CP-d4, and 340→114 for 4OHCP-d4-SCZ were selected for the internal standard (IS) corresponding to each analyte. The less abundant IS ions from 37Cl were used for CP-d4 and 4OHCP-d4-SCZ to overcome the cross-talk interference from the analytes. Under optimized conditions, retention times were 0.67 min for tepa and its IS, 2.50 min for thiotepa and its IS, 2.52 min for 4OHCP-SCZ and its IS, and 2.86 min for CP and its IS. Total run time was 5 min per sample. The calibration ranges were 2.5-2,000ng/mL for thiotepa and tepa, 20-10,000ng/mL for CP and 20-5,000 ng/mL for 4OHCP; Dilution integrity for samples above the calibration range was validated with 10-fold dilution for thiotepa/tepa and 20-fold dilution for CP/4OHCP. Recoveries ranged from 86.3-93.4% for thiotepa, 86.3-89.0% for tepa, 90.2-107% for CP, and 99.3-115% for 4OHCP-SCZ. The IS normalized matrix effect was within (100±7) % for all 4 analytes. Plasma samples at room temperature were stable for at least 60 hours for thiotepa, 6 days for tepa, and 24 hours for CP and 4OHCP-SCZ. Plasma samples for thiotepa/tepa were stable after 4 freeze-thaw cycles, and for CP/4OHCP-SCZ were stable after 3 freeze-thaw cycles. The assays were validated and applied to clinical studies requiring small sample volumes.

Determination of unbound piperaquine in human plasma by ultra-high performance liquid chromatography tandem mass spectrometry.

Piperaquine (PQ) is an antimalarial drug that is highly protein-bound. Variation in plasma protein contents may affect the pharmacokinetic (PK) exposure of unbound drug, leading to alteration of clinical outcomes. All published methods for determination of PQ in human plasma measure the total PQ including both bound and unbound PQ to plasma proteins. There is no published method for unbound PQ determination. Here we report an ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method for determination of PQ in human plasma filtrate prepared by filtering human plasma through Millipore Microcon® centrifugal filters (10k NMWL). The filter cup had to be treated with 5% benzalkonium chloride to reduce non-specific binding to the filter devices before filtration of plasma samples. Multiple reactions monitoring (MRM) of the ion pairs m/z 535/288 for PQ and m/z 541/294 for the internal standard (IS) was selected for quantification. When electrospray ionization (ESI+) was used, paradoxical matrix effect was observed despite the structure similarity of the deuterated IS: Ion suppression for PQ versus ion enhancement for the PQ-d6, even though they were closely eluted: 0.62 min versus 0.61 min. Separation was achieved on Evo C18 column (50 × 2.1 mm, 1.7 µm, Phenomenex Inc.) eluted with 10 mM NH4OH and MeCN. When atmospheric pressure chemical ionization in positive mode (APCI+) was used for ion source, matrix effect diminished. Separation was achieved on a PFP column (30 × 2.1 mm, 1.7 µm, Waters, Corp.) eluted with aqueous 20 mM ammonium formate 0.14% trifluoroacetic acid (A) and methanol-acetonitrile (4:1, v/v) containing 0.1% trifluoroacetic acid (B) at 0.8 mL/min flow rate in a gradient mode: 30-30-80-80-30-30%B (0-0.1-1.0-1.40-1.41-1.50 min). The retention time was 0.67 min for both PQ and the IS. The method was validated with a linear calibration range from 20 to 5,000 pg/mL and applied to clinical samples.

Piperaquine-Induced QTc Prolongation Decreases With Repeated Monthly Dihydroartemisinin-Piperaquine Dosing in Pregnant Ugandan Women.

BACKGROUND: Intermittent preventive treatment with monthly dihydroartemisinin-piperaquine (DHA-PQ) is highly effective at preventing both malaria during pregnancy and placental malaria. Piperaquine prolongs the corrected QT interval (QTc), and it is possible that repeated monthly dosing could lead to progressive QTc prolongation. Intensive characterization of the relationship between piperaquine concentration and QTc interval throughout pregnancy can inform effective, safe prevention guidelines. METHODS: Data were collected from a randomized controlled trial, where pregnant Ugandan women received malaria chemoprevention with monthly DHA-PQ (120/960 mg DHA/PQ; n = 373) or sulfadoxine-pyrimethamine (SP; 1500/75 mg; n = 375) during the second and third trimesters of pregnancy. Monthly trough piperaquine samples were collected throughout pregnancy, and pre- and postdose electrocardiograms were recorded at 20, 28, and 36 weeks' gestation in each woman. The pharmacokinetics-QTc relationship for piperaquine and QTc for SP were assessed using nonlinear mixed-effects modeling. RESULTS: A positive linear relationship between piperaquine concentration and Fridericia corrected QTc interval was identified. This relationship progressively decreased from a 4.42 to 3.28 to 2.13 millisecond increase per 100 ng/mL increase in piperaquine concentration at 20, 28, and 36 weeks' gestation, respectively. Furthermore, 61% (n = 183) of women had a smaller change in QTc at week 36 than week 20. Nine women given DHA-PQ had grade 3-4 cardiac adverse events. SP was not associated with any change in QTc. CONCLUSIONS: Repeated DHA-PQ dosing did not result in increased risk of QTc prolongation and the postdose QTc intervals progressively decreased. Monthly dosing of DHA-PQ in pregnant women carries minimal risk of QTc prolongation. CLINICAL TRIALS REGISTRATION: NCT02793622.

Identifying an optimal dihydroartemisinin-piperaquine dosing regimen for malaria prevention in young Ugandan children.

Intermittent preventive treatment (IPT) with dihydroartemisinin-piperaquine (DP) is highly protective against malaria in children, but is not standard in malaria-endemic countries. Optimal DP dosing regimens will maximize efficacy and reduce toxicity and resistance selection. We analyze piperaquine (PPQ) concentrations (n = 4573), malaria incidence data (n = 326), and P. falciparum drug resistance markers from a trial of children randomized to IPT with DP every 12 weeks (n = 184) or every 4 weeks (n = 96) from 2 to 24 months of age (NCT02163447). We use nonlinear mixed effects modeling to establish malaria protective PPQ levels and risk factors for suboptimal protection. Compared to DP every 12 weeks, DP every 4 weeks is associated with 95% protective efficacy (95% CI: 84-99%). A PPQ level of 15.4 ng/mL reduces the malaria hazard by 95%. Malnutrition reduces PPQ exposure. In simulations, we show that DP every 4 weeks is optimal across a range of transmission intensities, and age-based dosing improves malaria protection in young or malnourished children.

Long-term SARS-CoV-2-specific immune and inflammatory responses in individuals recovering from COVID-19 with and without post-acute symptoms.

We describe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cell responses, soluble markers of inflammation, and antibody levels and neutralization capacity longitudinally in 70 individuals with PCR-confirmed SARS-CoV-2 infection. Participants represent a spectrum of illness and recovery, including some with persistent viral shedding in saliva and many experiencing post-acute sequelae of SARS-CoV-2 infection (PASC). T cell responses remain stable for up to 9 months. Whereas the magnitude of early CD4+ T cell immune responses correlates with severity of initial infection, pre-existing lung disease is independently associated with higher long-term SARS-CoV-2-specific CD8+ T cell responses. Among participants with PASC 4 months following coronavirus disease 2019 (COVID-19) symptom onset, we observe a lower frequency of CD8+ T cells expressing CD107a, a marker of degranulation, in response to Nucleocapsid (N) peptide pool stimulation, and a more rapid decline in the frequency of N-specific interferon-γ-producing CD8+ T cells. Neutralizing antibody levels strongly correlate with SARS-CoV-2-specific CD4+ T cell responses.

Long-Term SARS-CoV-2-Specific Immune and Inflammatory Responses Across a Clinically Diverse Cohort of Individuals Recovering from COVID-19.

A detailed understanding of long-term SARS-CoV-2-specific T cell responses and their relationship to humoral immunity and markers of inflammation in diverse groups of individuals representing the spectrum of COVID-19 illness and recovery is urgently needed. Data are also lacking as to whether and how adaptive immune and inflammatory responses differ in individuals that experience persistent symptomatic sequelae months following acute infection compared to those with complete, rapid recovery. We measured SARS-CoV-2-specific T cell responses, soluble markers of inflammation, and antibody levels and neutralization capacity longitudinally up to 9 months following infection in a diverse group of 70 individuals with PCR-confirmed SARS-CoV-2 infection. The participants had varying degrees of initial disease severity and were enrolled in the northern California Long-term Impact of Infection with Novel Coronavirus (LIINC) cohort. Adaptive T cell responses remained remarkably stable in all participants across disease severity during the entire study interval. Whereas the magnitude of the early CD4+ T cell immune response is determined by the severity of initial infection (participants requiring hospitalization or intensive care), pre-existing lung disease was significantly associated with higher long-term SARS-CoV2-specific CD8+ T cell responses, independent of initial disease severity or age. Neutralizing antibody levels were strongly correlated with SARS-CoV-2-specific CD4+ T but not CD8+ T cell responses. Importantly, we did not identify substantial differences in long-term virus-specific T cell or antibody responses between participants with and without COVID-19-related symptoms that persist months after initial infection.

Development and validation of an LC-MS/MS method for determination of hydroxychloroquine, its two metabolites, and azithromycin in EDTA-treated human plasma.

BACKGROUND: Hydroxychloroquine (HCQ) and azithromycin (AZM) are antimalarial drugs recently reported to be active against severe acute respiratory syndrome coronavirus- 2 (SARS-CoV-2), which is causing the global COVID-19 pandemic. In an emergency response to the pandemic, we aimed to develop a quantitation method for HCQ, its metabolites desethylhydroxychloroquine (DHCQ) and bisdesethylchloroquine (BDCQ), and AZM in human plasma. METHODS: Liquid chromatography tandem mass spectrometry was used to develop the method. Samples (20 µL) are extracted by solid-phase extraction and injected onto the LC-MS/MS system equipped with a PFP column (2.0 × 50 mm, 3 µm). ESI+ and MRM are used for detection. Ion pairs m/z 336.1→247.1 for HCQ, 308.1→179.1 for DHCQ, 264.1→179.1 for BDCQ, and 749.6→591.6 for AZM are selected for quantification. The ion pairs m/z 342.1→253.1, 314.1→181.1, 270.1→181.1, and 754.6→596.6 are selected for the corresponding deuterated internal standards (IS) HCQ-d4, DHCQ-d4, BDCQ-d4, and AZM-d5. The less abundant IS ions from 37Cl were used to overcome the interference from the analytes. RESULTS: Under optimized conditions, retention times are 0.78 min for BDCQ, 0.79 min for DHCQ, 0.92 min for HCQ and 1.87 min for AZM. Total run time is 3.5 min per sample. The calibration ranges are 2-1000 ng/mL for HCQ and AZM, 1-500 ng/mL for DHCQ and 0.5-250 ng/mL for BDCQ; samples above the range are validated for up to 10-fold dilution. Recoveries of the method ranged from 88.9-94.4% for HCQ, 88.6-92.9% for DHCQ, 88.7-90.9% for BDCQ, and 98.6%-102% for AZM. The IS normalized matrix effect were within (100±10) % for all 4 analytes. Blood samples are stable for at least 6 hr at room temperature. Plasma samples are stable for at least 66 hr at room temperature, 38 days at -70°C, and 4 freeze-thaw cycles. CONCLUSIONS: An LC-MS/MS method for simultaneous quantitation of HCQ, DHCQ, BDCQ, and AZM in human plasma was developed and validated for clinical studies requiring fast turnaround time and small samples volume.

Pharmacokinetics and Safety of Zidovudine, Lamivudine, and Lopinavir/Ritonavir in HIV-infected Children With Severe Acute Malnutrition in Sub-Saharan Africa: IMPAACT Protocol P1092.

BACKGROUND: Severe acute malnutrition (SAM) may alter the pharmacokinetics (PK), efficacy, and safety of antiretroviral therapy. The phase IV study, IMPAACT P1092, compared PK, safety, and tolerability of zidovudine (ZDV), lamivudine (3TC), and lopinavir/ritonavir (LPV/r) in children with and without SAM. MATERIALS AND METHODS: Children living with HIV 6 to <36 months of age with or without World Health Organization (WHO)-defined SAM received ZDV, 3TC, and LPV/r syrup for 48 weeks according to WHO weight band dosing. Intensive PK sampling was performed at weeks 1, 12, and 24. Plasma drug concentrations were measured using liquid chromatography tandem mass spectrometry. Steady-state mean area under the curve (AUC0-12h) and clearance (CL/F) for each drug were compared. Grade ≥3 adverse events were compared between cohorts. RESULTS: Fifty-two children were enrolled across 5 sites in Africa with 44% (23/52) female, median age 19 months (Q1, Q3: 13, 25). Twenty-five children had SAM with entry median weight-for-height Z-score (WHZ) -3.4 (IQR -4.0, -3.0) and 27 non-SAM had median WHZ -1.0 (IQR -1.8, -0.1). No significant differences in mean AUC0-12h or CL/F were observed (P ≥ 0.09) except for lower 3TC AUC0-12h (GMR, 0.60; 95% CI, 0.4-1.0; P = 0.047) at week 12, higher ZDV AUC0-12h (GMR, 1.52; 1.2-2.0; P = 0.003) at week 24 in the SAM cohort compared with non-SAM cohort. Treatment-related grade ≥3 events did not differ significantly between cohorts (24.0% vs. 25.9%). CONCLUSION: PK and safety findings for ZDV, 3TC, and LPV/r support current WHO weight band dosing of syrup formulations in children with SAM.

Exposure to pesticides in utero impacts the fetal immune system and response to vaccination in infancy.

The use of pesticides to reduce mosquito vector populations is a cornerstone of global malaria control efforts, but the biological impact of most pesticides on human populations, including pregnant women and infants, is not known. Some pesticides, including carbamates, have been shown to perturb the human immune system. We measure the systemic absorption and immunologic effects of bendiocarb, a commonly used carbamate pesticide, following household spraying in a cohort of pregnant Ugandan women and their infants. We find that bendiocarb is present at high levels in maternal, umbilical cord, and infant plasma of individuals exposed during pregnancy, indicating that it is systemically absorbed and trans-placentally transferred to the fetus. Moreover, bendiocarb exposure is associated with numerous changes in fetal immune cell homeostasis and function, including a dose-dependent decrease in regulatory CD4 T cells, increased cytokine production, and inhibition of antigen-driven proliferation. Additionally, prenatal bendiocarb exposure is associated with higher post-vaccination measles titers at one year of age, suggesting that its impact on functional immunity may persist for many months after birth. These data indicate that in utero bendiocarb exposure has multiple previously unrecognized biological effects on the fetal immune system.

Everolimus, an mTORC1/2 inhibitor, in ART-suppressed individuals who received solid organ transplantation: A prospective study.

Pharmacologic inhibition of the mammalian target of rapamycin (mTOR) in the setting of renal transplantation has previously been associated with lower human immunodeficiency virus 1 (HIV-1) DNA burden, and in vitro studies suggest that mTOR inhibition may lead to HIV transcriptional silencing. Because prospective clinical trials are lacking, we conducted an open-label, single-arm study to determine the impact of the broad mTOR inhibitor, everolimus, on residual HIV burden, transcriptional gene expression profiles, and immune responses in HIV-infected adult solid organ transplant (SOT) recipients on antiretroviral therapy. Whereas everolimus therapy did not have an overall effect on cell-associated HIV-1 DNA and RNA levels in the entire cohort, participants who maintained everolimus time-averaged trough levels >5 ng/mL during the first 2 months of therapy had significantly lower RNA levels up to 6 months after the cessation of study drug. Time-averaged everolimus trough levels significantly correlated with greater inhibition of mTOR gene pathway transcriptional activity. Everolimus treatment also led to decreased PD-1 expression on certain T cell subsets. These data support the rationale for further study of the effects of mTOR inhibition on HIV transcriptional silencing in non-SOT populations, either alone or in combination with other strategies. Trial Registration: ClinicalTrials.gov NCT02429869.

Abnormal Levels of Some Biomarkers of Immune Activation Despite Very Early Treatment of Human Immunodeficiency Virus.

BACKGROUND: Despite early antiretroviral therapy (ART), ART-suppressed people with human immunodeficiency virus (HIV) (PWH) remain at higher risk for infections and infection-related cancers than the general population. The immunologic pathways that remain abnormal in this setting, potentially contributing to these complications, are unclear. METHODS: ART-suppressed PWH and HIV-negative controls, all cytomegalovirus seropositive and enriched for HIV risk factors, were sampled from an influenza vaccine responsiveness study. PWH were stratified by timing of ART initiation (within 6 months of infection [early ART] vs later) and nadir CD4+ T-cell count among later initiators. Between-group differences in kynurenine-tryptophan (KT) ratio, interferon-inducible protein 10, soluble CD14 and CD163, soluble tumor necrosis factor receptor 2, interleukin 6, and soluble urokinase plasminogen activator receptor were assessed after confounder adjustment. RESULTS: Most participants (92%) were male, reflecting the demographics of early-ART initiators in San Francisco. Most biomarkers were higher among later-ART initiators. Participants in the early-ART group achieved near-normal soluble tumor necrosis factor receptor 2, interleukin 6, and soluble urokinase plasminogen activator receptor levels, but substantially higher KT ratio than those without HIV after confounder adjustment (P = .008). Soluble CD14, soluble CD163, and interferon-inducible protein 10 trended similarly. CONCLUSIONS: While early-ART initiators restore near-normal levels of many inflammatory markers, the kynurenine pathway of tryptophan catabolism remains abnormally high. Because this pathway confers adaptive immune defects and predicts tuberculosis and cancer progression, this it may contribute to persistent risks of these complications in this setting.