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Study Design: Retrospective review of the Healthcare Cost and Utilization Project National Inpatient Sample, 2000 to 2013., Objective: To determine the proportion of spinal epidural abscess (SEA) cases that were related to injection drug use (IDU) and to compare length of stay, leaving against medical advice, paralysis, cauda equina syndrome, radiculitis, and in-hospital mortality between SEA cases with and without IDU., Summary of Background Data: The US opioid epidemic impacts all aspects of healthcare, including spinal surgeons. Although injection drug use (IDU) is a risk factor for spinal epidural abscess (SEA), IDU among SEA patients and its effect on clinical outcomes is not well understood., Methods: Cases aged 15 to 64 with principal diagnosis of SEA were classified as IDU-related (IDU-SEA) or non-IDU-related (non-IDU-SEA) using International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM) codes for illicit drug use and hepatitis C. We determined the proportion of SEA patients with IDU and compared length of stay, leaving against medical advice, paralysis, cauda equina syndrome, radiculitis, and in-hospital mortality between IDU-SEA and non-IDU-SEA patients., Results: From 2000 to 2013, there were 20,425 admissions with a principal diagnosis of SEA (95% confidence interval (CI), 19,281-21,568); 19.1% were associated with IDU (95% CI, 17.7%-20.5%). The proportion of white IDU-SEA cases increased by 2.4 percentage points annually (95% CI, 1.4-3.4). After adjusting for age, sex, and race, IDU-SEA patients stayed a mean of 6.7 more days in the hospital (95% CI, 5.1-8.2) and were 4.8 times more likely to leave against medical advice (95% CI, 2.9-8.0). Mean hospital charges for IDU-SEA patients were $31,603 higher (95% CI: $20,721-$42,485). Patients with IDU-SEA were less likely to have cauda equina syndrome (adjusted odds ratio, 0.48, 95% CI, 0.26-0.87)., Conclusion: IDU-SEA patients stay in the hospital longer and more often leave against medical advice. Providers and hospitals may benefit from exploring how to better facilitate completion of inpatient treatment and achieve superior outcomes., Level of Evidence: 3.

People with opioid use disorder (OUD) have worse hospital outcomes and higher healthcare costs. There are rising reports of people with OUD also using other classes of drugs, however patterns of substance use have not been evaluated for differential effects on hospital outcomes. We performed a data-analysis of the Healthcare Utilization Project's National Readmissions Database, examining the effects of patterns of substance use, age, gender, and diagnosis on the outcomes of Against Medical Advice (AMA) discharges and 30-day readmissions. About one-third of the patients with OUD who were admitted to the hospital had at least one additional substance use disorder (SUD). Thirteen percent of persons with OUD were discharged AMA, and 12% were readmitted to the hospital within 30 days of discharge. Compared to people with OUD alone, people who used stimulants had increased odds of AMA discharge (aOR 1.83 (CI 1.73, 1.96)) and 30-day readmission (aOR 1.30 (95% CI 1.23, 1.37)). Multiple concomitant substance use disorders were associated with increased odds of AMA discharge and 30-day readmission. Conclusions: People with OUD have high rates of both AMA discharges and 30 day-readmissions, and there is a layered effect of increasing co-occurring SUDs leading to worse hospitalization outcomes. The heterogeneity of drug use patterns needs to be considered when developing strategies to improve health care outcomes for people with substance use disorder., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Fueled by the burgeoning opioid epidemic, valve surgeries for substance use disorder-related infective endocarditis (SUD-IE) are increasing. The impact of substance use disorder on postvalve replacement morbidity needs further investigation., Methods: We queried The Society of Thoracic Surgeons Adult Cardiac Surgery Database for all valve surgeries for infective endocarditis at Tufts Medical Center (2002-2016) and collected demographic and disease-related data, including timing of mortality subclassified as short-term (<6 months including operative), midterm (6 months to 5 years), and extended-term (>5 years). Patients with documentation of illicit drug use before the operation were considered to have SUD-IE. Deaths were confirmed through review of medical record and matching with the Massachusetts Vital Statistics Database. We performed univariate and multivariate proportional hazard regressions examining the impact of substance use disorder mortality in people who received a valve replacement., Results: In the cohort of 228 patients, 80 (35%) had SUD-IE. Eight-six people (38%) died, and overall mortality was higher in people with SUD-IE compared with non-SUD-IE (48% vs 32%, P = .025). SUD-IE was associated with a higher risk of overall mortality (adjusted hazard ratio, 2.41; 95% confidence interval, 1.38-4.20; P = .002). Although the difference between short-term or extended-term mortality was not significant, SUD-IE was associated with increased frequency of midterm mortality (53% vs 31%, P = .003)., Conclusions: Our data reflect high rates of postvalve surgery morbidity and mortality in people with SUD-IE at a tertiary care center. The midterm postoperative period is a vulnerable period for people with SUD-IE worthy of further investigation., (Copyright © 2020 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

Hepatitis B and hepatitis C (HCV) prevalence are higher in people on hemodialysis (HD) than the general population. Through implementation of prevention interventions including vaccines, serologic screening, and post-exposure management, transmissions linked to HD have decreased dramatically. In this manuscript, we review epidemiology of viral hepatitis, summarize current screening and vaccine recommendations, and appraise the available data about efforts to decrease incidence within HD facilities, including isolation of people with viral hepatitis within HD units. Also included is a discussion of the highly effective all-oral HCV treatment options and treatment for HCV in people awaiting kidney transplant., (© 2020 Wiley Periodicals LLC.)

Background: In the midst of an opioid epidemic, health care workers are encountering an increasing number of patients who have opioid use disorder in addition to complex social, behavioral and medical issues. Of all the clinicians in the hospital, nurses spend the most time with hospitalized patients who have opioid use disorder, yet there has been little research exploring their experiences in caring for this population. The objective of this study was to assess the attitudes, perceptions, and training needs of nurses in the inpatient setting when caring for patients who have opioid use disorder., Methods: One-on-one in-depth interviews were conducted with nurses working at a large academic medical center in Boston, MA, using a semi-structured interview guide. Nurses were recruited via email notifications and subsequent snowball sampling. Interviews were recorded, transcribed and analyzed using a grounded theory approach., Results: Data from in-depth interviews with 22 nurses were grouped into six themes: (1) stigma, (2) assessing & treating pain, (3) feelings of burn out, (4) communication between providers, (5) safety & security, and (6) opportunities for change. These themes were organized within four ecological levels of the Socio-Ecological Model: I) societal context, II) hospital environment, III) interpersonal interactions, and IV) individual factors. Nurses were cognizant of the struggles that patients who have opioid use disorder confront during hospitalization such as pain, withdrawal and stigma, and elaborated on how these challenges translate to professional and emotional strain among nurses. Nurses offered recommendations by which the hospital could streamline care for this population, including expanded role support for nurses and more structured policies regarding care for patients who present with a comorbid opioid use disorder., Conclusion: Our results highlight the need for the development of programs targeting both organizational culture and the inpatient nurse quality of life to ultimately enhance quality of care for patients who present with opioid use disorder., Competing Interests: The authors have declared that no competing interests exist.

Background: Infective endocarditis (IE) often requires surgical intervention. An increasingly common cause of IE is injection drug use (IDU-IE). There is conflicting evidence on whether postoperative mortality differs between people with IDU-IE and people with IE from etiologies other than injection drug use (non-IDU-IE). In this manuscript, we compare short-term postoperative mortality in IDU-IE vs non-IDU-IE through systematic review and meta-analysis., Methods: The review was conducted according to the Cochrane Handbook for Systematic Reviews of Interventions and the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. Publication databases were queried for key terms included in articles up to September 2017. Randomized controlled trials, prospective cohorts, or retrospective cohorts that reported on 30-day mortality or in-hospital/operative mortality following valve surgery and that compared outcomes between IDU-IE and non-IDU-IE were included., Results: Thirteen studies with 1593 patients (n = 341 [21.4%] IDU-IE) were included in the meta-analysis. IDU-IE patients more frequently had tricuspid valve infection, Staphylococcus infection, and heart failure before surgery. Meta-analysis revealed no statistically significant difference in 30-day postsurgical mortality or in-hospital mortality between the 2 groups., Conclusions: Despite differing preoperative clinical characteristics, early postoperative mortality does not differ between IDU-IE and non-IDU-IE patients who undergo valve surgery. Future research on long-term outcomes following valve replacement is needed to identify opportunities for improved healthcare delivery with IDU-IE., (Published by Oxford University Press for the Infectious Diseases Society of America 2018.)

Hepatitis C virus (HCV) is highly prevalent in incarcerated populations. The high cost of HCV therapy places a major burden on correctional system healthcare budgets, but the burden of untreated HCV is not known. We investigated the economic impact of HCV through comparison of length of stay (LOS), frequency of 30-day readmission, and costs of hospitalizations in inmates with and without HCV using a 2004-2014 administrative claims database. Inmates with HCV had longer LOS, higher frequency of 30-day readmission, and increased cost of hospitalizations. Costs were higher in inmates with HCV even without advanced liver disease and in inmates with HIV/HCV compared to HCV alone. We conclude that although HCV treatment may not avert all of the observed increases in hospitalization, modest reductions in hospital utilization with HCV cure could help offset treatment costs. Policy discussions on HCV treatment in corrections should be informed by the costs of untreated HCV infection.

Background: The United States has a growing opioid epidemic impacting all aspects of health care including orthopaedic surgery. Septic arthritis of the knee is a condition commonly encountered by orthopaedic surgeons related to opioid and injection drug use (IDU). Changes in the frequency of hospitalizations for IDU-related septic arthritis and differences in septic arthritis patient outcomes according to IDU status in the setting of the burgeoning opioid epidemic are unknown., Questions/purposes: (1) What proportion of patients with septic arthritis of the knee use injection drugs? (2) Are there any differences in complications, reoperations, length of stay, and leaving against medical advice among patients with septic arthritis of the knee with and without IDU? (3) What are the age and racial trends in IDU-related septic arthritis of the knee from 2000 to 2013?, Methods: The Healthcare Cost and Utilization Project, Nationwide Inpatient Sample database of years 2000 to 2013 was utilized for patients between ages 15 and 64 years with a principal discharge diagnosis of native septic arthritis of the lower leg, the vast majority of which represents the knee. The Nationwide Inpatient Sample is the largest publicly available healthcare database in the United States that can show nationally representative clinical trends and outcomes. Septic arthritis was classified as related or unrelated to IDU based on previously published algorithms using billing codes. Patients with IDU-related septic arthritis were more likely to be black or Hispanic, younger, and use Medicare, Medicaid, or self-payment as their primary payment method. The yearly proportion of patients with septic arthritis who used injection drugs was determined. Hospitalization outcomes including length of stay, leaving against medical advice, number of procedures, and mortality rates were compared after adjusting for age, gender, and race in multivariable regression analyses. The yearly change in proportion of IDU-related septic arthritis in each age, race, and gender group was compared over the study period., Results: The proportion of patients with IDU-related septic arthritis increased from 5% in 2000 to 11% in 2013. After adjusting for age, gender, and race, patients with IDU-related septic arthritis were more likely to die during hospitalization (adjusted odds ratio [AOR], 2.86; 95% confidence interval [CI], 1.51-5.39; p < 0.001) and undergo repeat arthroscopic (AOR, 1.24; 95% CI, 1.06-1.45; p = 0.007) or open irrigation and débridement (AOR, 1.68; 95% CI, 1.28-2.19; p < 0.001). Patients with IDU-related septic arthritis were more likely to leave against medical advice (AOR, 7.13; 95% CI, 5.56-9.15; p < 0.001) and also had an additional 5 days in length of stay (95% CI, 4.1-5.5; p < 0.001) on average compared with patients with septic arthritis unrelated to IDU. There was an increasing proportion of patients with IDU-related septic arthritis who were aged 15 to 34 years and 55 to 64 years from 2000 to 2013., Conclusions: IDU is increasingly the cause of septic knee admissions and is associated with higher rates of mortality, reoperations, resource utilization, and leaving against medical advice. Orthopaedic surgeons must adequately screen for IDU among patients with septic arthritis and monitor them closely for reoperation with a low threshold to reaspirate a knee in the postoperative period. Future studies should determine the current use and potential benefits of a multidisciplinary approach, including addiction specialists, to aid in the management of the increasing number of these patients., Level of Evidence: Level III, therapeutic study.

Background: Abscesses commonly occur among people who inject drugs (PWID). However, whether the risks are comparable between males and females, and the impact of sex work on abscess risk is unclear. The goal of this study was to examine the contemporary associations of gender and sex work with the risk of abscesses in PWID., Methods: Combining data from two cross-sectional studies conducted in the Greater Boston Area with people at risk for HIV and hepatitis C virus (HCV), we used the following inclusion criteria: age 18-45 years and report of illicit or non-prescription drug injection within the 30 days prior to the survey. Information on demographics, injection-mediated risks, and sexual behaviors was collected using Audio Computer-Assisted Self-Interview Software. Multivariable logistic regressions were used to model associations., Results: The study sample included 298 people including 30% were female. Females were more likely than men to report sex work (28% vs. 16%, p = .012) and abscess during their lifetime (55% vs. 37% p = .004). Among the females, engaging in sex work increased by >5-fold the odds of reporting abscesses [Adjusted odds ratio 5.42; 95% CI: 1.27, 23.10]. There was no association between sex work and abscesses among men., Discussion: We found a female-specific association between sex work, injection drug use, and abscesses among PWID. Although the cross-sectional designs precluded causal inferences, longitudinal studies could enhance understanding of gender-associated risks for abscesses and inform the development of harm reduction interventions., (Published by Elsevier B.V.)

Shigellosis has emerged as a nontraditional sexually transmitted infection with high rates among men who have sex with men. Although anecdotal evidence has surfaced regarding shigellosis clustering among men who have sex with men in Massachusetts, little scientific research documents these occurrences. We present the first spatial distribution of shigellosis across Massachusetts.

There are several barriers to annual hepatitis C virus antibody (HCVAb) testing, including lack of provider knowledge of the changing HCV epidemic and provider underestimation of a patient's risk. We identified low rates of testing for HCVAb in people living with human immunodeficiency virus (HIV) in our outpatient HIV Infectious Diseases clinic, and we developed a quality improvement project to increase rates of HCVAb screening.

In the absence of other epidemiological exposures, shigellosis in men who have sex with men (MSM) suggests engagement in high-risk sexual encounters. Infection and diagnosis of shigellosis in MSM presents a potentially overlooked opportunity to discuss the availability of pre-exposure prophylaxis medications to prevent incident HIV infections., (© The Author(s) 2016.)

People who inject drugs (PWID) are at risk for infective endocarditis (IE). Hospitalization rates related to misuse of prescription opioids and heroin have increased in recent years, but there are no recent investigations into rates of hospitalizations from injection drug use-related IE (IDU-IE). Using the Health Care and Utilization Project National Inpatient Sample (HCUP-NIS) dataset, we found that the proportion of IE hospitalizations from IDU-IE increased from 7% to 12.1% between 2000 and 2013. Over this time period, we detected a significant increase in the percentages of IDU-IE hospitalizations among 15- to 34-year-olds (27.1%-42.0%; P < .001) and among whites (40.2%-68.9%; P < .001). Female gender was less common when examining all the IDU-IE (40.9%), but it was more common in the 15- to 34-year-old age group (53%). Our findings suggest that the demographics of inpatients hospitalized with IDU-IE are shifting to reflect younger PWID who are more likely to be white and female than previously reported. Future studies to investigate risk behaviors associated with IDU-IE and targeted harm reduction strategies are needed to avoid further increases in morbidity and mortality in this rapidly growing population of young PWID.

Background.  The incidence of hepatitis C virus (HCV) infection is increasing in human immunodeficiency virus (HIV)-positive men who have sex with men (MSM). New guidelines recommend annual screening for HCV, similar to recommendations for syphilis screening with rapid plasma reagin (RPR). Methods.  This study compares the frequency of repeat HCV antibody (Ab) testing to repeat RPR testing in a retrospective chart review of 359 HCVAb-negative people living with HIV (PLWH) observed in an Infectious Diseases clinic. Patients were classified into risk groups based on sexual risk factors. Results.  Although 85% of PLWH had repeat syphilis screening, less than two thirds had repeat HCVAb screening. The MSM status was associated with increased HCVAb and RPR testing (adjusted odds ratio, 2.6 and 5.9, respectively). Seven persons had incident HCV infection: 3 were MSM, and 4 had symptoms or abnormal laboratory results to prompt testing. Conclusions.  Failure to find incident HCV infection in PLWH represents missed opportunities to cure HCV infection and prevent progressive liver disease. Further quality improvement studies are necessary to develop physician-focused interventions to increase HCV screening rates in PLWH.

Illicit drug use can result in a wide range of medical complications. As the availability, synthesis, and popularity of illicit drugs evolve over time, new syndromes associated with their use may mimic infections. Some of these symptoms are anticipated drug effects, and others are complications of adulterants mixed with drugs or complications from the method of using drugs. Some illicit drugs are associated with rare infections, which are difficult to diagnosis with standard microbiological techniques. The goal of this review is to orient a wide range of clinicians-including general practitioners, emergency medicine providers, and infectious diseases specialists-to complications of illicit drug use that may be underrecognized. Improving awareness of infectious and noninfectious complications of illicit drug can expedite diagnosis and medical treatment of persons who use drugs and facilitate targeted harm reduction counseling to prevent future complications., (© The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Disease caused by Powassan virus (POWV), a tick-borne flavivirus, ranges from asymptomatic to severe neurologic compromise and death. Two cases of POWV meningoencephalitis in New York, USA, highlight diagnostic techniques, neurologic outcomes, and the effect of POWV on communities to which it is endemic.

Health care workers (HCW) rely on Internet-based medical resources to obtain current evidence-based clinical guidelines. While such resources are being used with increasing frequency in developed countries, they have not been broadly employed in resource-limited settings (RLS). To investigate the feasibility and the types of medical content used by HCW in RLS, we studied one Internet-based medical resource used in the United States, (UpToDate©) in four hospitals in Africa. Health care workers were trained on how to use this resource and surveyed over a six month study period. Automated topic tracking found that HCW searched a variety of medical topics. About 78% of HCW reported daily or weekly use of the Internet-based medical resource and 70% felt the tool was very useful for teaching. All users report the tool increased their clinical knowledge. This descriptive study suggests that Internet-based medical information resources are feasible in RLS and are accessed for a broad variety of medical topics. Supplemental content providing guidance when minimal diagnostic and therapeutic options exist could increase its relevance in RLS. With increased availability, Internet-based medical information could enhance current global health care initiatives to improve providers' knowledge, clinical practice and potentially patient outcomes.

Background: CD8+ T cells impact control of viral infections by direct elimination of infected cells and secretion of a number of soluble factors. In HIV-1 infection, persistent HIV-1 specific IFN-gamma+ CD8+ T cell responses are detected in the setting of disease progression, consistent with functional impairment in vivo. Recent data suggest that impaired maturation, as defined by the lineage markers CD45RA and CCR7, may contribute to a lack of immune control by these responses., Methodology/principal Findings: We investigated the maturation phenotype of epitope-specific CD8+ T cell responses directed against HIV-1 in 42 chronically infected, untreated individuals, 22 of whom were "Controllers" (median 1140 RNA copies/ml plasma, range<50 to 2520), and 20 "progressors" of whom had advanced disease and high viral loads (median 135,500 RNA copies/ml plasma, range 12100 to >750000). Evaluation of a mean of 5 epitopes per person revealed that terminally differentiated CD8+ T cells directed against HIV-1 are more often seen in HIV-1 Controllers (16/22; 73%) compared to HIV-1 progressors (7/20; 35%)(p = 0.015), but the maturation state of epitope-specific responses within a given individual was quite variable. Maturation phenotype was independent of the HLA restriction or the specificity of a given CD8+ T cell response and individual epitopes associated with slow disease progression were not more likely to be terminally differentiated., Conclusions/significance: These data indicate that although full maturation of epitope-specific CD8+ T cell responses is associated with viral control, the maturation status of HIV-1 specific CD8+ T cell responses within a given individual are quite heterogeneous, suggesting epitope-specific influences on CD8+ T cell function.

Background: Hepatitis C virus (HCV)-specific T cell responses are critical for spontaneous resolution of HCV viremia. Here we examined the effect of a lymphotropic virus, HIV-1, on the ability of coinfected patients to maintain spontaneous control of HCV infection., Methods and Findings: We measured T cell responsiveness by lymphoproliferation and interferon-gamma ELISPOT in a large cohort of HCV-infected individuals with and without HIV infection. Among 47 HCV/HIV-1-coinfected individuals, spontaneous control of HCV was associated with more frequent HCV-specific lymphoproliferative (LP) responses (35%) compared to coinfected persons who exhibited chronic HCV viremia (7%, p = 0.016), but less frequent compared to HCV controllers who were not HIV infected (86%, p = 0.003). Preservation of HCV-specific LP responses in coinfected individuals was associated with a higher nadir CD4 count (r(2) = 0.45, p < 0.001) and the presence and magnitude of the HCV-specific CD8(+) T cell interferon-gamma response (p = 0.0014). During long-term follow-up, recurrence of HCV viremia occurred in six of 25 coinfected individuals with prior control of HCV, but in 0 of 16 HIV-1-negative HCV controllers (p = 0.03, log rank test). In these six individuals with recurrent HCV viremia, the magnitude of HCV viremia following recurrence inversely correlated with the CD4 count at time of breakthrough (r = -0.94, p = 0.017)., Conclusions: These results indicate that HIV infection impairs the immune response to HCV-including in persons who have cleared HCV infection-and that HIV-1-infected individuals with spontaneous control of HCV remain at significant risk for a second episode of HCV viremia. These findings highlight the need for repeat viral RNA testing of apparent controllers of HCV infection in the setting of HIV-1 coinfection and provide a possible explanation for the higher rate of HCV persistence observed in this population.

Despite limited data supporting the superiority of dominant over subdominant responses, immunodominant epitopes represent the preferred vaccine candidates. To address the function of subdominant responses in human immunodeficiency virus infection, we analyzed cytotoxic T lymphocyte responses restricted by HLA-B*1503, a rare allele in a cohort infected with clade B, although common in one infected with clade C. HLA-B*1503 was associated with reduced viral loads in the clade B cohort but not the clade C cohort, although both shared the immunodominant response. Clade B viral control was associated with responses to several subdominant cytotoxic T lymphocyte epitopes, whereas their clade C variants were less well recognized. These data suggest that subdominant responses can contribute to in vivo viral control and that high HLA allele frequencies may drive the elimination of subdominant yet effective epitopes from circulating viral populations.

A vigorous hepatitis C virus (HCV)-specific Th cell response is regarded as essential to the immunological control of HCV viremia. The aim of this study was to comprehensively define the breadth and specificity of dominant HCV-specific CD4(+) T cell epitopes in large cohorts of subjects with chronic and spontaneously resolved HCV viremia. Following in vitro stimulation of PBMC, HCV-specific cell cultures from each subject were screened with an overlapping panel of synthetic 20-mer peptides spanning the entire HCV polyprotein. Of 22 subjects who spontaneously controlled HCV viremia, all recognized at least one of a group of six epitopes situated within the nonstructural (NS) proteins NS3, NS4, and NS5, each of which was detected by >30% of subjects, but most subjects recognized additional, more heterogeneous specificities. In contrast, none of the most frequently targeted epitopes was detected by >5% of persons with chronic infection. The most frequently recognized peptides showed promiscuous binding to multiple HLA-DR molecules in in vitro binding assays and were restricted by different HLA-DR molecules in functional assays in different persons. These data demonstrate that predominant CD4(+) T cell epitopes in persons with resolved HCV infection are preferentially located in the nonstructural proteins and are immunogenic in the context of multiple class II molecules. This comprehensive characterization of CD4(+) T cell epitopes in resolved HCV infection provides important information to facilitate studies of immunopathogenesis and HCV vaccine design and evaluation.

Several HLA class I alleles have been associated with slow human immunodeficiency virus (HIV) disease progression, supporting the important role HLA class I-restricted cytotoxic T lymphocytes (CTL) play in controlling HIV infection. HLA-B63, the serological marker for the closely related HLA-B*1516 and HLA-B*1517 alleles, shares the epitope binding motif of HLA-B57 and HLA-B58, two alleles that have been associated with slow HIV disease progression. We investigated whether HIV-infected individuals who express HLA-B63 generate CTL responses that are comparable in breadth and specificity to those of HLA-B57/58-positive subjects and whether HLA-B63-positive individuals would also present with lower viral set points than the general population. The data show that HLA-B63-positive individuals indeed mounted responses to previously identified HLA-B57-restricted epitopes as well as towards novel, HLA-B63-restricted CTL targets that, in turn, can be presented by HLA-B57 and HLA-B58. HLA-B63-positive subjects generated these responses early in acute HIV infection and were able to control HIV replication in the absence of antiretroviral treatment with a median viral load of 3,280 RNA copies/ml. The data support an important role of the presented epitope in mediating relative control of HIV replication and help to better define immune correlates of controlled HIV infection.

CD8(+) T-cell responses are an essential antiviral host defense in persistent viral infections, and their sustained effectiveness is thought to be critically dependent on CD4(+) T-helper cells. To determine the relationship between HIV-1-induced CD4(+) T-cell depletion and hepatitis C virus (HCV)-specific CD8(+) T-cell responses during viral persistence, we studied 103 persons positive for HCV, 74 coinfected with HIV-1. CD8(+) T-cell responses to the entire HCV polyprotein were determined by using an interferon-gamma enzyme-linked immunospot (ELISpot) assay. Although HIV-1 infection by itself was not associated with a diminished HCV-specific response, HIV-1-associated CD4(+) depletion was associated with significantly lower HCV-specific CD8(+) T cells (R = 0.48, P < .0001). In contrast, declining CD4(+) counts over the same range were not associated with diminished Epstein-Barr virus (EBV)- (R = 0.19, P = .31) or HIV-1-specific (R = -0.13, P = .60) CD8(+) T-cell responses in persons infected with all viruses. These data indicate that frequencies of circulating HCV-specific CD8(+) T-cell responses are sensitive to absolute CD4(+) T-cell counts and provide a possible explanation for the accelerated HCV disease course in persons coinfected with HIV-1 and HCV.

China is a region of the world with a rapidly spreading HIV-1 epidemic. Studies providing insights into HIV-1 pathogenesis in infected Chinese are urgently needed to support the design and testing of an effective HIV-1 vaccine for this population. HIV-1-specific T cell responses were characterized in 32 HIV-1-infected individuals of Chinese origin and compared to 34 infected caucasians using 410 overlapping peptides spanning the entire HIV-1 clade B consensus sequence in an IFN-gamma ELISpot assay. All HIV-1 proteins were targeted with similar frequency in both populations and all study subjects recognized at least one overlapping peptide. HIV-1-specific T cell responses clustered in seven different regions of the HIV-1 genome in the Chinese cohort and in nine different regions in the caucasian cohort. The dominant HLA class I alleles expressed in the two populations differed significantly, and differences in epitope clustering pattern were shown to be influenced by differences in class I alleles that restrict immunodominant epitopes. These studies demonstrate that the clustering of HIV-1-specific T cell responses is influenced by the genetic HLA class I background in the study populations. The design and testing of candidate vaccines to fight the rapidly growing HIV-1 epidemic must therefore take the HLA genetics of the population into account as specific regions of the virus can be expected to be differentially targeted in ethnically diverse populations.

Although there is increasing evidence that virus-specific cytotoxic-T-lymphocyte (CTL) responses play an important role in the control of human immunodeficiency virus (HIV) replication in vivo, only scarce CTL data are available for the ethnic populations currently most affected by the epidemic. In this study, we examined the CD8(+)-T-cell responses in African-American, Caucasian, Hispanic, and Caribbean populations in which clade B virus dominates and analyzed the potential factors influencing immune recognition. Total HIV-specific CD8(+)-T-cell responses were determined by enzyme-linked immunospot assays in 150 HIV-infected individuals by using a clade B consensus sequence peptide set spanning all HIV proteins. A total of 88% of the 410 tested peptides were recognized, and Nef- and Gag-specific responses dominated the total response for each ethnicity in terms of both breadth and magnitude. Three dominantly targeted regions within these proteins that were recognized by >90% of individuals in each ethnicity were identified. Overall, the total breadth and magnitude of CD8(+)-T-cell responses correlated with individuals' CD4 counts but not with viral loads. The frequency of recognition for each peptide was highly correlated with the relative conservation of the peptide sequence, the presence of predicted immunoproteasomal cleavage sites within the C-terminal half of the peptide, and a reduced frequency of amino acids that impair binding of optimal epitopes to the restricting class I molecules. The present study thus identifies factors that contribute to the immunogenicity of these highly targeted and relatively conserved sequences in HIV that may represent promising vaccine candidates for ethnically heterogeneous populations.

CD8 T-cell responses are thought to be crucial for control of viremia in human immunodeficiency virus (HIV) infection but ultimately fail to control viremia in most infected persons. Studies in acute infection have demonstrated strong CD8-mediated selection pressure and evolution of mutations conferring escape from recognition, but the ability of CD8 T-cell responses that persist in late-stage infection to recognize viruses present in vivo has not been determined. Therefore, we studied 24 subjects with advanced HIV disease (median viral load = 142,000 copies/ml; median CD4 count = 71/ micro l) and determined HIV-1-specific CD8 T-cell responses to all expressed viral proteins using overlapping peptides by gamma interferon Elispot assay. Chronic-stage virus was sequenced to evaluate autologous sequences within Gag epitopes, and functional avidity of detected responses was determined. In these subjects, the median number of epitopic regions targeted was 13 (range, 2 to 39) and the median cumulative magnitude of CD8 T-cell responses was 5,760 spot-forming cells/10(6) peripheral blood mononuclear cells (range, 185 to 24,700). On average six (range, one to 8) proteins were targeted. For 89% of evaluated CD8 T-cell responses, the autologous viral sequence was predicted to be well recognized by these responses and the majority of analyzed optimal epitopes were recognized with medium to high functional avidity by the contemporary CD8 T cells. Withdrawal of antigen by highly active antiretroviral therapy led to a significant decline both in breadth (P = 0.032) and magnitude (P = 0.0098) of these CD8 T-cell responses, providing further evidence that these responses had been driven by recognition of autologous virus. These results indicate that strong, broadly directed, and high-avidity gamma-interferon-positive CD8 T-cells directed at autologous virus persist in late disease stages, and the absence of mutations within viral epitopes indicates a lack of strong selection pressure mediated by these responses. These data imply functional impairment of CD8 T-cell responses in late-stage infection that may not be reflected by gamma interferon-based screening techniques.

Increasing efforts are directed towards the development of effective vaccines through induction of virus-specific T cell responses. Although emerging data indicate a significant role of these cells in determining viral set point in infections such as HIV, there is as yet no consensus as to the best methods for assaying the breadth of these responses. In this study, we used sensitive interferon gamma-based intracellular cytokine staining (ICS) and Elispot assays to determine the optimal overlapping peptide set to screen for these responses. Twenty persons with established HIV infection were studied, focusing on responses to the highly immunogenic Nef protein. Six different HIV-1 Nef peptide sets were used, ranging in length from 15 to 20 amino acids (aa), in overlap from 10 to 11 amino acids, and derived from two different B clade sequences. A total of 54 CD8 T cell responses to Nef peptides were found in this cohort, of which only 12 were detected using previously defined Nef optimal epitopes. No single peptide set detected all responses. Though there was a trend of the shorter peptides detecting more CD8 T cell responses than the 20 amino acid long peptides and longer peptides detecting more CD4 T cell responses, neither was statistically significant. There was no difference between an overlap of 10 or 11 amino acids. All responses detected with the six different sets of overlapping peptides were towards the more highly conserved regions of Nef. We conclude that peptides ranging from 15 to 20 amino acids yield similar results in IFN-gamma-based Elispot and ICS assays, and that all are likely to underestimate the true breadth of responses to a given reference strain of virus.

Cellular immune responses play a critical role in the control of human immunodeficiency virus type 1 (HIV-1); however, the breadth of these responses at the single-epitope level has not been comprehensively assessed. We therefore screened peripheral blood mononuclear cells (PBMC) from 57 individuals at different stages of HIV-1 infection for virus-specific T-cell responses using a matrix of 504 overlapping peptides spanning all expressed HIV-1 proteins in a gamma interferon-enzyme-linked immunospot (Elispot) assay. HIV-1-specific T-cell responses were detectable in all study subjects, with a median of 14 individual epitopic regions targeted per person (range, 2 to 42), and all 14 HIV-1 protein subunits were recognized. HIV-1 p24-Gag and Nef contained the highest epitope density and were also the most frequently recognized HIV-1 proteins. The total magnitude of the HIV-1-specific response ranged from 280 to 25,860 spot-forming cells (SFC)/10(6) PBMC (median, 4,245) among all study participants. However, the number of epitopic regions targeted, the protein subunits recognized, and the total magnitude of HIV-1-specific responses varied significantly among the tested individuals, with the strongest and broadest responses detectable in individuals with untreated chronic HIV-1 infection. Neither the breadth nor the magnitude of the total HIV-1-specific CD8+-T-cell responses correlated with plasma viral load. We conclude that a peptide matrix-based Elispot assay allows for rapid, sensitive, specific, and efficient assessment of cellular immune responses directed against the entire expressed HIV-1 genome. These data also suggest that the impact of T-cell responses on control of viral replication cannot be explained by the mere quantification of the magnitude and breadth of the CD8+-T-cell response, even if a comprehensive pan-genome screening approach is applied.

Vigorous virus-specific CD4+ T-helper-cell responses are associated with successful control of hepatitis C virus (HCV) and other human viral infections, but the breadth and specificity of responses associated with viral containment have not been defined. To address this we evaluated the HCV-specific CD4+ T-helper-cell response in HCV antibody-positive persons who lack detectable plasma viremia, and compared this response to that in persons with chronic HCV infection. Peripheral blood mononuclear cells were stimulated with HCV proteins, followed by measurement of HCV-specific CD4+ T-cell responses to a comprehensive set of overlapping HCV peptides by intracellular gamma interferon production. In three persons with resolved HCV infection studied in detail, 13 to 14 epitopes were targeted, but none was recognized by all three. The 37 defined epitopes were predominantly distributed among the HCV proteins core, NS3, NS4, and NS5. In an expanded analysis of responses to these proteins in persons with resolved infection, an average of 10 epitopes was targeted, whereas in persons with chronic viremia never was more than one epitope targeted (P < 0.001). This comprehensive analysis of the breadth and specificity of HCV-specific T-helper-cell responses indicates that up to 14 viral epitopes can be simultaneously targeted by circulating virus-specific CD4+ T helper cells in a controlled human viral infection. Moreover, these data provide important parameters for evaluation of candidate HCV vaccines, and provide rationale for immunotherapy in chronic HCV infection.