T-cell acute lymphoblastic leukemia in patients 1-45 years treated with the pediatric NOPHO ALL2008 protocol.

The NOPHO ALL2008 is a population-based study using an unmodified pediatric protocol in patients 1-45 years of age with acute lymphoblastic leukemia. Patients with T-ALL were given a traditional pediatric scheme if fast responding (minimal residual disease (MRD) < 0.1% day 29), or intensive block-based chemotherapy if slow responding (MRD > 0.1% day 29). Both treatment arms included pediatric doses of high-dose methotrexate and asparaginase. If MRD ≥ 5% on day 29 or ≥0.1% after consolidation, patients were assigned to allogeneic hematopoietic stem cell transplantation. The 5-year overall survival of the 278 T-ALL patients was 0.75 (95% CI 0.69-0.81), being 0.82 (0.74-0.88) for patients 1.0-9.9 years, 0.76 (0.66-0.86) for those 10.0-17.9 years, and 0.65 (0.55-0.75) for the older patients. The risk of death in first remission was significantly higher in adults (12%) compared with the 1-9 years group (4%). The MRD responses in the three age groups were similar, and only a nonsignificant increase in relapse risk was found in adults. In conclusion, an unmodified pediatric protocol in patients 1-45 years is effective in all age groups. The traditional pediatric treatment schedule was safe for all patients, but the intensive block therapy led to a high toxic death rate in adults.

The EKiTE network (epidemiology in kidney transplantation - a European validated database): an initiative epidemiological and translational European collaborative research.

BACKGROUND: Kidney transplantation is considered to be the treatment of choice for people with end-stage renal disease (ESRD). However, due to the shortage of available organs and the increase in the ESRD prevalence in Europe, it is essential to improve transplantation outcomes by studying the related prognostic factors. Today, there is no European registry collecting data to perform such clinical epidemiology studies. MAIN BODY: Entitled EKiTE, for European cohort for Kidney Transplantation Epidemiology, this prospective and multicentric cohort includes patients from Spanish (Barcelona), Belgian (Leuven), Norwegian (Oslo) and French (Paris Necker, Lyon, Nantes, Nancy, Montpellier, Nice and Paris Saint Louis) transplantation centers and currently contains 13,394 adult recipients of kidney (only) transplantation from 2005 and updated annually. A large set of parameters collected from transplantation until graft failure or death with numbers of post-transplantation outcomes. The long-term follow-up and the collected data enable a wide range of possible survival and longitudinal analyses. CONCLUSION: EKiTE is a multicentric cohort aiming to better assess the natural history of the ESRD in European kidney transplant recipients and perform benchmarking of clinical practices. The data are available for clinical epidemiology studies and open for external investigators upon request to the scientific council. Short-term perspectives are to extend EKITE network to other European countries and collect additional parameters in respect of the common thesaurus.

Contraceptive Choices and Counseling in Norwegian Female Renal Transplant Recipients.

BACKGROUND: There are major gaps in the understanding of sexual and reproductive health in female renal transplant recipients. METHODS: In this Norwegian multicenter retrospective observational study, 118 female renal transplant recipients aged 22 to 49 years responded to a questionnaire on fertility, contraceptive use, and pregnancy. RESULTS: More than one-third (37%) of patients reported that they did not receive advice on contraceptive methods from health care personnel in the early post-transplant phase. These women used effective contraceptive methods less often. Nearly half of the patients (45%) reported that they had not received any advice on timing of conception after transplant. From 95 pregnancies after renal transplant, 52 (55%) resulted in live births. CONCLUSION: Counseling on contraceptive methods should be part of standard care in conjunction with transplantation. More than one-third of young female renal transplant recipients of reproductive age could not recall having received advice from health care personnel about contraceptive use, and nearly half of the patients did not receive preconceptional advice after transplant. Although the current study does not discriminate between lack of advice and recall bias, the findings signal the need for improved counseling on female sexual and reproductive health after renal transplant.

Results of NOPHO ALL2008 treatment for patients aged 1-45 years with acute lymphoblastic leukemia.

Adults with acute lymphoblastic leukemia (ALL) do worse than children. From 7/2008 to 12/2014, Nordic and Baltic centers treated 1509 consecutive patients aged 1-45 years with Philadelphia chromosome-negative ALL according to the NOPHO ALL2008 without cranial irradiation. Overall, 1022 patients were of age 1-9 years (A), 266 were 10-17 years (B) and 221 were 18-45 years (C). Sixteen patients (three adults) died during induction. All others achieved remission after induction or 1-3 intensive blocks. Subsequently, 45 patients (12 adults) died, 122 patients relapsed (32 adults) with a median time to relapse of 1.6 years and 13 (no adult) developed a second malignancy. Median follow-up time was 4.6 years. Among the three age groups, older patients more often had higher risk ALL due to T-ALL (32%/25%/9%, P<0.001), KMT2A rearrangements (6%/5%/3%, P<0.001) and higher day 29 residual leukemia for B-lineage (P<0.001), but not T-ALL (P=0.53). Event-free survival rates (pEFS5y) were 89±1% (A), 80±3% (B) and 74±4% (C) with significant differences only for non-high risk groups. Except for thrombosis, pancreatitis and osteonecrosis, the risk of 19 specified toxicities was not enhanced by age above 10 years. In conclusion, a pediatric-based protocol is tolerable and effective for young adults, despite their increased frequency of higher risk features.

Outcomes in Pancreas Transplantation With Exocrine Drainage Through a Duodenoduodenostomy Versus Duodenojejunostomy.

Until recently, pancreas transplantation has mostly been performed with exocrine drainage via duodenojejunostomy (DJ). Since 2012, DJ was substituted with duodenoduodenostomy (DD) in our hospital, allowing endoscopic access for biopsies. This study assessed safety profiles with DD versus DJ procedures and clinical outcomes with the DD technique in pancreas transplantation. DD patients (n = 117; 62 simultaneous pancreas-kidney [SPKDD ] and 55 pancreas transplantation alone [PTADD ] with median follow-up 2.2 years) were compared with DJ patients (n = 179; 167 SPKDJ and 12 PTADJ ) transplanted in the period 1998-2012 (pre-DD era). Postoperative bleeding and pancreas graft vein thrombosis requiring relaparotomy occurred in 17% and 9% of DD patients versus 10% (p = 0.077) and 6% (p = 0.21) in DJ patients, respectively. Pancreas graft rejection rates were still higher in PTADD patients versus SPKDD patients (p = 0.003). Hazard ratio (HR) for graft loss was 2.25 (95% CI 1.00, 5.05; p = 0.049) in PTADD versus SPKDD recipients. In conclusion, compared with the DJ procedure, the DD procedure did not reduce postoperative surgical complications requiring relaparatomy or improve clinical outcomes after pancreas transplantation despite serial pancreatic biopsies for rejection surveillance. It remains to be seen whether better rejection monitoring in DD patients translates into improved long-term pancreas graft survival.

Collectin Liver 1 and Collectin Kidney 1 of the Lectin Complement Pathway Are Associated With Mortality After Kidney Transplantation.

Kidney transplanted patients still have significantly higher mortality compared with the general population. The innate immune system may play an important role during periods, with suppression of the adaptive immune system. In the present study, two soluble pattern recognition molecules of the innate immune system were investigated, collectin liver 1 (CL-L1) and collectin kidney 1 (CL-K1). Potential associations of their pretransplant levels and long-term graft and recipient survival were examined. The levels of CL-L1 and CL-K1 were measured at the time of transplantation in 382 patients (≥17 years) transplanted in 2000-2001. The cohort was subsequently followed until December 31, 2014. Data on patient and graft survival were obtained from the Norwegian Renal Registry. Both high CL-L1 (≥376 ng/mL) and high CL-K1 (≥304 ng/mL) levels were significantly associated with overall mortality in multivariate Cox analyses with hazard ration (HR) 1.50, 95% confidence interval (CI) 1.09-2.07, p = 0.013 and HR 1.43, 95% CI 1.02-1.99, p = 0.038, respectively. Moreover, high CL-K1 levels were significantly associated with cardiovascular mortality. No association between measured biomarkers and death-censored graft loss was found. Finally, there was a significant correlation between these two collectins, r = 0.83 (95% CI 0.80-0.86). In conclusion, CL-L1 and CL-K1 were significantly associated with mortality in kidney transplant recipients.

Plasma levels of marine n-3 fatty acids and cardiovascular risk markers in renal transplant recipients.

BACKGROUND/OBJECTIVES: Cardiovascular (CV) disease is the leading cause of death after renal transplantation. Marine n-3 polyunsaturated fatty acids (PUFAs) exert potential cardio-protective metabolic effects and might reduce CV morbidity and mortality in renal transplant recipients (RTRs). SUBJECTS/METHODS: In this cross-sectional study of 1990 Norwegian RTRs, transplanted between 1999 and 2011, associations between plasma phospholipid marine n-3 PUFA levels and various CV risk markers at 10 weeks after transplant were evaluated. RESULTS: Higher plasma marine n-3 PUFA levels were associated with lower resting heart rate (rHR), lower fasting plasma glucose (fPG) levels, lower plasma triglyceride levels and higher plasma high-density lipoprotein (HDL) cholesterol levels. Plasma levels of eicosapentaenoic acid, but not docosahexaenoic acid, showed a positive association with plasma HDL cholesterol levels. Plasma marine n-3 PUFA levels were not associated with plasma low-density lipoprotein cholesterol levels, pulse wave velocity or systolic and diastolic blood pressure. A negative association between plasma marine n-3 PUFA levels and CV mortality was weakened by additional adjustment for plasma triglyceride levels and rHR. The ratio of n-6 to n-3 PUFAs showed similar associations with CV risk markers as absolute plasma marine n-3 PUFA levels. CONCLUSIONS: This is the first study in RTRs showing that marine n-3 PUFAs are negatively associated with rHR and fPG in addition to beneficial effects on plasma HDL cholesterol and triglyceride levels. Especially, effects on autonomic nervous function and triglyceride metabolism might contribute to explain the lower CV mortality risk with higher plasma marine n-3 PUFA levels previously shown in this cohort.

Serum Calcification Propensity Is a Strong and Independent Determinant of Cardiac and All-Cause Mortality in Kidney Transplant Recipients.

Calcification of the vasculature is associated with cardiovascular disease and death in kidney transplant recipients. A novel functional blood test measures calcification propensity by quantifying the transformation time (T50 ) from primary to secondary calciprotein particles. Accelerated T50 indicates a diminished ability of serum to resist calcification. We measured T50 in 1435 patients 10 weeks after kidney transplantation during 2000-2003 (first era) and 2009-2012 (second era). Aortic pulse wave velocity (APWV) was measured at week 10 and after 1 year in 589 patients from the second era. Accelerated T50 was associated with diabetes, deceased donor, first transplant, rejection, stronger immunosuppression, first era, higher serum phosphate and lower albumin. T50 was not associated with progression of APWV. During a median follow-up of 5.1 years, 283 patients died, 70 from myocardial infarction, cardiac failure or sudden death. In Cox regression models, accelerated T50 was strongly and independently associated with both all-cause and cardiac mortality, low versus high T50 quartile: hazard ratio 1.60 (95% confidence interval [CI] 1.00-2.57), ptrend = 0.03, and 3.60 (95% CI 1.10-11.83), ptrend = 0.02, respectively. In conclusion, calcification propensity (T50 ) was strongly associated with all-cause and cardiac mortality of kidney transplant recipients, potentially via a cardiac nonAPWV-related pathway. Whether therapeutic improvement of T50 improves outcome awaits clarification in a randomized trial.

Complexity of Host Micro-RNA Response to Cytomegalovirus Reactivation After Organ Transplantation.

Human (Homo sapiens) micro-RNAs (hsa-miRNAs) regulate virus and host-gene translation, but the biological impact in patients with human cytomegalovirus (hCMV) infection is not well defined in a clinically relevant model. First, we compared hsa-miRNA expression profiles in peripheral blood mononuclear cells from 35 transplant recipients with and without CMV viremia by using a microarray chip covering 847 hsa-miRNAs. This approach demonstrated a set of 142 differentially expressed hsa-miRNAs. Next, we examined the effect of each of these miRNAs on viral growth by using human fibroblasts (human foreskin fibroblast-1) infected with the hCMV Towne strain, identifying a subset of proviral and antiviral hsa-miRNAs. miRNA-target prediction software indicated potential binding sites within the hCMV genome (e.g., hCMV-UL52 and -UL100 [UL = unique long]) and host-genes (e.g., interleukin-1 receptor, IRF1). Luciferase-expressing plasmid constructs and immunoblotting confirmed several predicted miRNA targets. Finally, we determined the expression of selected proviral and antiviral hsa-miRNAs in 242 transplant recipients with hCMV-viremia. We measured hsa-miRNAs before and after antiviral therapy and correlated hsa-miRNA expression levels to hCMV-replication dynamics. One of six antiviral hsa-miRNAs showed a significant increase during treatment, concurrent with viral decline. In contrast, six of eight proviral hsa-miRNAs showed a decrease during viral decline. Our results indicate that a complex and multitargeted hsa-miRNA response occurs during CMV replication in immunosuppressed patients. This study provides mechanistic insight and potential novel biomarkers for CMV replication.

Hcmv-miR-UL22A-5p: A Biomarker in Transplantation With Broad Impact on Host Gene Expression and Potential Immunological Implications.

Cytomegalovirus (CMV) encodes multiple microRNAs. While these have been partially characterized in vitro, their relevance to clinical CMV infection has not been evaluated. We analyzed samples from a cohort of solid organ transplant patients with CMV disease (n = 245) for viral microRNA expression. Several CMV microRNAs were readily detectable in patients with CMV disease in variable relative abundance. Expression level generally correlated with DNA viral load and the absence of viral microRNA was associated with faster viral clearance. Detection of hcmv-miR-UL22A-5p at baseline independently predicted the recurrence of CMV viremia upon discontinuation of antiviral therapy (OR 3.024, 95% CI: 1.35-6.8; p = 0.007). A combination of direct mRNA targeting by the microRNA and indirect modulation of gene expression involving isoforms of the transcriptional regulator C-MYC may be responsible for the broad effects seen in the association of gene transcripts with the RNA-induced silencing complex and in global protein expression upon hcmv-miR-UL22A-5p transfection. This novel study of in vivo viral microRNA expression profiles provides unique insight into the complexity of clinical CMV infection following transplantation. We provide evidence that viral microRNAs may have complex effects on gene expression and be associated with specific virologic and clinical outcomes, and thus could be further evaluated as biomarkers.

Secreted Wnt antagonists during eradication of cytomegalovirus infection in solid organ transplant recipients.

We evaluated secreted wingless (Wnt) modulators during cytomegalovirus (CMV) infection in solid organ transplant recipients (SOTr). The major findings were: (i) Plasma levels of Dickkopf-1 (DKK-1) were significantly lower in patients with CMV DNAemia above lower level of quantification at baseline. (ii) Receiver operating characteristic analysis indicated that low DKK-1 and increased secreted frizzled related protein-3 levels were predictors of poor virological outcomes during follow-up. Our findings demonstrate an imbalanced pattern of circulating secreted Wnt modulators in SOTr with poor virological outcomes following treatment for CMV disease, and may suggest a role for dysregulated Wnt signaling on viral pathogenesis during CMV infection.

Activated CD8(+) T cells and NKT cells in BAL fluid improve diagnostic accuracy in sarcoidosis.

PURPOSE: The clinical diagnosis of pulmonary sarcoidosis is based on the presence of noncaseating granulomas in an appropriate clinical setting with either bilateral hilar adenopathy and/or parenchymal infiltrates. Lymphocytosis with an increased CD4/CD8 T cell ratio in bronchoalveolar lavage fluid is supportive. We evaluated the diagnostic accuracy of a predictive binary logistic regression model in sarcoidosis based on sex, age, and bronchoalveolar lavage fluid cell profile with and without the inclusion of HLA-DR(+) CD8(+) T cells and natural killer T-cell fractions. METHODS: A retrospective analysis of differential cell counts and lymphocyte phenotypes by flow cytometry in bronchoalveolar lavage was performed in 183 patients investigated for possible diffuse parenchymal lung disease. A logistic regression model with age, sex, lymphocyte fraction, eosinophils, and CD4/CD8 ratio in bronchoalveolar lavage fluid (basic model) was compared with a final model, which also included fractions of HLA-DR(+) CD8(+) T cells and natural killer T cells. Diagnostic accuracy of the two models was assessed by receiver operating characteristic (ROC) curves. RESULTS: The area under the ROC curve for the basic and final model was 0.898 [95 % confidence interval (CI) 0.852-0.945] and 0.937 (95 % CI 0.902-0.972), respectively, p = 0.008. CONCLUSIONS: Assessment of HLA-DR(+) CD8(+) T cell and natural killer T-cell fractions may improve diagnostic accuracy and further strengthen the importance of bronchoalveolar lavage in the diagnostic workup of sarcoidosis.

Empty iron stores in children and young adults--the diagnostic accuracy of MCV, MCH, and MCHC.

INTRODUCTION: Erythrocyte mean cell volume (MCV) is used clinically to classify anemia, and normal values may be used to exclude iron deficiency. We have studied the diagnostic accuracy of MCV and the related measures mean cell hemoglobin (MCH) and mean cell hemoglobin concentration (MCHC) in diagnosing empty iron stores in children and young adults. METHODS: Diagnostic accuracy of MCV, MCH, and MCHC was studied by ROC curve analysis in 6443 ambulant patients aged 0.5-25 years, of which 476 were anemic. In all patients, blood hemoglobin, MCV, MCH, and serum ferritin were measured in specimens sampled at the same time. MCHC was calculated as MCH divided by MCV. The gold standard of empty iron stores was s-ferritin <10, 15, or 20 µg/L. The cutoff limit of MCV giving 90% sensitivity in diagnosing serum ferritin <15 µg/L was constructed using quantile regression. RESULTS: Generally, MCH was slightly more accurate than MCV and MCHC. In the whole study population, the area under the ROC curve was 0.68-0.93 for MCV, 0.73-0.96 for MCH, and 0.68-0.87 for MCHC; and 0.70-0.86, 0.71-0.89, and 0.68-0.88, respectively, in the anemic subpopulation. At the cutoff limits of MCV giving a sensitivity of 90% at all ages in anemic patients, the specificity was about 50%. CONCLUSION: Mean cell hemoglobin, MCH, and MCHC are only moderately accurate in diagnosing empty iron stores in children and young adults, and normal values of these tests do not exclude empty iron stores in anemic patients.

New algorithm for valganciclovir dosing in pediatric solid organ transplant recipients.

CMV infections are common after SOT. v-GCV is increasingly used in children. The aim of this study was to evaluate presently used dosing algorithms. Data from 104 pediatric SOT recipients (kidney, liver, and heart) aged 0.3-16.9 yr and receiving v-GCV once a day were used for model development and validation with the Pmetrics package for R. Monte Carlo simulations were performed to compare the probability of a GCV AUC 40-60 mg*h/L with the different algorithms across a range of ages, weights, and GFRs. GCV pharmacokinetics was well described by the non-parametric model. Clearance was dependent on GFR and Cockcroft-Gault estimates improved the model fit over Schwartz. Simulations showed that our new algorithm, where v-GCV dose is: Weight [kg]*(0.07*GFR [mL/min]+k), where k = 5 for GFR ≤ 30 mL/min, k = 10 for GFR > 30 mL/min and weight > 30 kg and k = 15 for GFR > 30 mL/min and weight ≤ 30 kg, outperformed the other algorithms. Thirty-three percent of all patients achieve an exposure above and 21% within the therapeutic window. We propose a simple algorithm for initial v-GCV dosing that standardizes plasma drug exposure better than current algorithms. Subsequent TDM is strongly suggested to achieve individual drug levels within the therapeutic window.

Evaluation of an In Silico PBPK Post-Bariatric Surgery Model through Simulating Oral Drug Bioavailability of Atorvastatin and Cyclosporine.

An increasing prevalence of morbid obesity has led to dramatic increases in the number of bariatric surgeries performed. Altered gastrointestinal physiology following surgery can be associated with modified oral drug bioavailability (Foral). In the absence of clinical data, an indication of changes to Foral via systems pharmacology models would be of value in adjusting dose levels after surgery. A previously developed virtual "post-bariatric surgery" population was evaluated through mimicking clinical investigations on cyclosporine and atorvastatin after bariatric surgery. Cyclosporine simulations displayed a reduced fraction absorbed through gut wall (fa) and Foral after surgery, consistent with reported observations. Simulated atorvastatin Foral postsurgery was broadly reflective of observed data with indications of counteracting interplay between reduced fa and an increased fraction escaping gut wall metabolism (FG). Inability to fully recover observed atorvastatin exposure after biliopancreatic diversion with duodenal switch highlights the current gap regarding the knowledge of associated biological changes.CPT: Pharmacometrics & Systems Pharmacology (2013) 2, e47; doi:10.1038/psp.2013.23; advance online publication 12 June 2013.

Impact of OATP1B1, MDR1, and CYP3A4 expression in liver and intestine on interpatient pharmacokinetic variability of atorvastatin in obese subjects.

Individual variability in expression and function of organic anion-transporting polypeptide 1B1 (OATP1B1), multidrug resistance protein 1 (MDR1), and/or cytochrome P450 3A4 (CYP3A4) may impact the clinical response of many drugs. We investigated the correlation between expression of these proteins and pharmacokinetics of atorvastatin, a substrate of all three, in 21 obese patients with paired biopsies from liver and intestinal segments. The patients were also screened for the SLCO1B1 c.521T→C variant alleles. Approximately 30% (r(2) = 0.28) of the variation in oral clearance (CL/F) of atorvastatin was explained by hepatic OATP1B1 protein expression (P = 0.041). Patients carrying the SLCO1B1 c.521C variant allele (homozygous, n = 4; heterozygous, n = 2) exhibited 45% lower CL/F of atorvastatin than the c.521TT carriers (P = 0.067). No association between hepatic and intestinal expression of MDR1 or CYP3A4 and atorvastatin pharmacokinetics was found (P > 0.149). In conclusion, this study suggests that OATP1B1 phenotype is more important than CYP3A4 and MDR1 phenotypes for the individual pharmacokinetic variability of atorvastatin.

A 1-year randomized, double-blind, placebo-controlled study of intravenous ibandronate on bone loss following renal transplantation.

The clinical profile of ibandronate as add-on to calcitriol and calcium was studied in this double-blind, placebo-controlled trial of 129 renal transplant recipients with early stable renal function (≤ 28 days posttransplantation, GFR ≥ 30 mL/min). Patients were randomized to receive i.v. ibandronate 3 mg or i.v. placebo every 3 months for 12 months on top of oral calcitriol 0.25 mcg/day and calcium 500 mg b.i.d. At baseline, 10 weeks and 12 months bone mineral density (BMD) and biochemical markers of bone turnover were measured. The primary endpoint, relative change in BMD for the lumbar spine from baseline to 12 months was not different, +1.5% for ibandronate versus +0.5% for placebo (p = 0.28). Ibandronate demonstrated a significant improvement of BMD in total femur, +1.3% versus -0.5% (p = 0.01) and in the ultradistal radius, +0.6% versus -1.9% (p = 0.039). Bone formation markers were reduced by ibandronate, whereas the bone resorption marker, NTX, was reduced in both groups. Calcium and calcitriol supplementation alone showed an excellent efficacy and safety profile, virtually maintaining BMD without any loss over 12 months after renal transplantation, whereas adding ibandronate significantly improved BMD in total femur and ultradistal radius, and also suppressed biomarkers of bone turnover. Ibandronate was also well tolerated.

Analysis and clinical correlation of genetic variation in cytomegalovirus.

BACKGROUND: Cytomegalovirus (CMV) displays genetic polymorphisms in multiple genes, which may result in important virulence differences. Glycoprotein N (gN) and immediate early 1 (IE1) are key viral genes and immune targets. We aimed to characterize the molecular epidemiology of gN and IE1 genotypes in organ transplant patients with CMV disease in the context of clinical and virologic endpoints. METHODS: A total of 240 patients with CMV disease had genotyping analysis by nested polymerase chain reaction assays and sequencing using blood samples obtained at disease onset. Results were correlated with viral clearance kinetics and recurrence. RESULTS: Complex patterns of gN and IE1 genotypes were present with no clear genetic linkages. No single genotype of IE1 or gN was associated with poorer outcome. For example, different gN or IE1 genotypes had comparable baseline viral load, clearance half-lives, time to clearance, and rates of virologic recurrence. Mixed infection was present at IE1 in 15.8% and gN in 21.9%, but analysis of a single gene was insufficient to detect all mixed infections. Infections caused by multiple strains, as opposed to single strains, were associated with higher baseline viral loads (P = 0.011), delayed viral clearance (P = 0.033), and higher rates of virologic recurrence (P = 0.008). CONCLUSIONS: Genetic diversity in CMV is complex. Specific gN or IE subtypes do not seem to affect in vivo viral virulence patterns in single-strain infections. Mixed infections demonstrate associations with virologic outcomes that single-strain infections do not.