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Bictegravir is a potent integrase strand-transfer inhibitor (INSTI) with a high genetic barrier to resistance. Bictegravir, coformulated with emtricitabine and tenofovir alafenamide, is recommended by key European and US HIV treatment guidelines as the preferred single-tablet regimen for adults and adolescents. The aim of this study was to assess the pharmacokinetics, safety, and efficacy of switching to this regimen in virologically suppressed children and adolescents with HIV.In this single-arm, open-label trial, we enrolled virologically suppressed children and adolescents (aged 6 to18 years) with HIV at 22 hospital clinics in South Africa, Thailand, Uganda, and the USA. Eligible participants had a bodyweight of at least 25 kg, were virologically suppressed (HIV-1 RNA50 copies per mL) on a stable ART regimen for at least 6 months before screening, had a CD4 count of at least 200 cells per μL, and an estimated glomerular filtration rate of at least 90 mL/min per 1·73 mBetween Sept 29, 2016 and Feb 16, 2018, we enrolled 102 participants. 100 participants received bictegravir, emtricitabine, and tenofovir alafenamide (cohort 1 [adolescents aged 12 to18 years], n=50; cohort 2 [children aged 6 to12 years], n=50). The mean bictegravir AUCIn adolescents and children with HIV, the bictegravir, emtricitabine, and tenofovir alafenamide single-tablet regimen was well tolerated and maintained virological suppression. Our data support the treatment of HIV in adolescents and children with this single-tablet regimen. At present, the single-tablet regimen is recommended as first-line treatment in the USA for adolescents and as an alternative regimen in children and has the potential to represent an important regimen in the paediatric population.Gilead Sciences.

We tested a novel dot survey methodology at our clinic that provides sexual health services to youth ages 13 to 24. We conducted two interactive dot surveys to assess their feasibility and acceptability while gaining insight into patients' attitudes about mental health.We adapted a dot survey approach to assess youths' familiarity with mental health and attitudes toward related services. We also assessed their attitudes toward participating in this survey method. All patients with scheduled appointments were eligible to participate. Participants used dot stickers to indicate their responses on survey posters displayed in the waiting room.Three hundred patients participated between June and September 2021 (150 participants/survey). About 95% of participants liked seeing others' responses to the dot surveys, and over 70% reported that the surveys made them think more about mental health. Over 90% would participate in future dot surveys at the clinic. Survey items with the most consensus among participants included that 74.5% "really agree" youth face barriers to accessing mental health services (n = 141, mean = 4.61, standard deviation = 0.79) and 87.1% "really agree" primary care providers should ask youth about their mental health (n = 139, mean = 4.81, standard deviation = 0.59).The dot surveys were effective at assessing patients' attitudes about mental health and feasible to conduct in our waiting room. Results confirmed that this survey method was well received among patients. Dot surveys can be adapted by other clinical settings to engage youth regarding their health-related attitudes.

Young men who have sex with men (YMSM) living with HIV experience challenges with retention in care, which negatively affects viral suppression. To address this, researchers piloted Bijou, a program designed to provide health education through electronically delivered behavior and risk reduction modules. Participants were 29 YMSM aged 19-24 living with HIV from the southeastern US. Participants completed pre, post, and 3-month follow-up (3MFU) surveys assessing knowledge, intervention acceptability, satisfaction, self-efficacy, ehealth literacy, and usability. Findings revealed significant improvement in knowledge and e-health literacy from pre-test to post-test but lost significance at 3MFU. Self-efficacy scores did not show significant differences from pre-test to post-test or 3MFU. Participants who completed all modules considered Bijou usable and acceptable; however, many did not complete the program. Findings suggest a need for adaptations to promote knowledge retention, e-health literacy, engagement over time, and research with a larger, more representative sample.

In adults, data support the utility and acceptance of home HIV testing; however, in youth, particularly in the US, this has not been well studied. In this exploratory study, we surveyed Tampa Bay youth aged 16−27 and attending sexual health clinics between 1 June and 31 June 2018 (n = 133) regarding attitudes and perceptions towards HIV self-testing. While most indicated the clinic over home when asked for preferred testing location, study population and subgroup analysis demonstrated a positive response (agree) to Likert-scale questions regarding the use of home HIV self-testing kits and negative responses (strongly disagree) to “would not use self-testing kit”. There was a significant difference between genders in testing location preference (p = 0.031) for those respondents that specified gender (n = 123), with males more likely to prefer home testing than females. This study suggests an openness of youth towards HIV home testing that could help to expand the number of youth aware of their HIV status.

Approximately 22% of HIV diagnoses in 2015 occurred among youth aged 13-24. Much is known about the risk factors and psychopathology present in youth living with HIV (YLWH), however, relatively little is known about resiliency in this population. The current study sought to assess factors related to resilience and vulnerability among YLWH as well as the impact of psychosocial factors on these constructs using existing clinical data from an integrated care clinic serving YLWH in the southeastern United States. Data included findings from mental health screeners administered as part of the standard protocol of care for youth aged 13-24 including information about anxiety (GAD-7), post-traumatic stress disorder (PC-PTSD), depression (PHQ-A or PHQ-9), substance use (CRAFFT), and medication adherence (BEHKA-HIV Action subscale) as well as viral load and demographic variables. Hierarchical linear regression was used to determine factors related to biological (viral load) and behavioral indicators of resilience and vulnerability (BEHKA-HIV Action subscale and CRAFFT). Results showed that anxiety was a significant covariate of both biological and behavioral indicators of resilience while gender was a significant factor associated with behavioral indicators of vulnerability. None of the psychological or demographic factors examined in this study were associated with substance use, a behavioral indicator of vulnerability and resilience. Our results support the need for clinicians to screen for and monitor anxiety symptoms among YLWH in integrated care settings in an effort to promote resilience and minimize vulnerability. Practical, evidence-based strategies should be applied in clinical settings to address medication adherence and anxiety among YLWH.

Rates of depression, anxiety, and posttraumatic stress disorder (PTSD) are greater in youth living with HIV (YLWH) than in the general population. Currently, depression screening guidelines exist f...

Background: Bictegravir a potent integrase strand transfer inhibitor (INSTI) with a high barrier to resistance, coformulated with emtricitabine and tenofovir alafenamide, is a guidelines-recommended and preferred single-tablet regimen for adults and adolescents. The aim of this study was to evaluate the pharmacokinetics, safety, and efficacy of switching to this regimen in virologically suppressed children and adolescents living with HIV. Methods: In this prospective, multi-country, 48-week, Phase 2/3, single-arm, open-label trial (ClinicalTrials.gov, number NCT02881320), virologically suppressed children and adolescents (6 to

Health literacy influences HIV treatment for youth and, thus, is a research priority. We explored health knowledge and self-reported adherence, as indicators of health literacy, among youth living with HIV (YLWH) and the association between health literacy and health outcomes. A total of 102 YLWH ages 13-25 years participated in the study. Participants completed the Brief Estimate of Health Knowledge and Action-HIV Version; CD4 T-cell counts and viral loads were extracted from participant medical records. Participants had a moderate amount of HIV knowledge, and most reported taking their medications under most conditions. Decreasing action scores were statistically associated with an increased likelihood of having a detectable viral load. Health literacy is an important factor that should be addressed by practitioners working with YLWH. More research is needed to determine the best way to measure and improve health literacy.

Youth living with HIV (YLWH) face significant mental health problems, namely depression, anxiety, and PTSD with rates of these disorders higher than in the general population. This study explored the relationship between symptoms of depression, anxiety, and PTSD and biological markers among a sample of 145 YLWH ages 13-25 years. Participants completed the Center for Epidemiologic Studies Depression Scale (CES-D), Generalized Anxiety Disorder-7 Item Scale (GAD-7), and Primary Care-Posttraumatic Stress Disorder Screen (PC-PTSD). Biological markers included CD4 count and viral load (VL) abstracted from medical records. Findings revealed a relationship between depression and anxiety and CD4 count as well as anxiety and VL. The relationship between depression and anxiety and CD4 count and anxiety and VL was moderated by transmission mode (i.e., behavioral versus perinatal). For youth perinatally infected, greater psychological symptoms of depression and anxiety were associated with a decline in CD4 count and increase in VL, but this was not true for youth with behaviorally acquired HIV. These findings point to the need for individualized mental health prevention and intervention services for YLWH.

Background Depressive symptoms are well documented among people living with HIV and some evidence suggests that youth living with HIV (YLWH) are more affected than their adult counterparts. Therefore, screening for depression is imperative among YLWH to ensure optimal health. The objective of this study is to compare the utility of the Center for Epidemiological Studies-Depression (CES-D) and the Patient Health Questionnaire (PHQ) as depression screeners in an integrated care setting serving YLWH in the southeastern United States. Methods As a part of standard care, the CES-D and the PHQ were administered to YLWH. A Retrospective review of patient records was conducted. Using receiver operating characteristic (ROC) curve analysis and reports from mental health providers, researchers compared the utility of the screeners. Results The sample consisted of 121 cases from 2017. Youth ranged in age from 12–25 (M = 20.68, SD = 2.75). Most were Black/African American (59.5%) males (56.2%) who acquired HIV behaviorally (51.2%). Sexual orientation was nearly evenly split between heterosexual (37.2%) and homosexual (34.7%). The CES-D demonstrated higher specificity and sensitivity for identifying clinical depression, yet, this was not significantly different from the PHQ, p = .09. Limitations Generalizability of findings may be limited as the study sample included youth from a single integrated care setting. Conclusion Both the PHQ and the CES-D demonstrate utility for depression screening among YLWH. However, the PHQ may be preferable for use within a clinical setting.

The Xpert Flu+RSV Xpress Assay is a fast, automated in vitro diagnostic test for qualitative detection and differentiation of influenza A and B viruses and respiratory syncytial virus (RSV) performed on the Cepheid GeneXpert Xpress System. The objective of this study was to establish performance characteristics of the Xpert Flu+RSV Xpress Assay compared to those of the Prodesse ProFlu+ real-time reverse transcription-PCR (RT-PCR) assay (ProFlu+) for the detection of influenza A and B viruses as well as RSV in a Clinical Laboratory Improvement Amendments (CLIA)-waived (CW) setting. Overall, the assay, using fresh and frozen nasopharyngeal (NP) swabs, demonstrated high concordance with results of the ProFlu+ assay in the combined CW and non-CW settings with positive percent agreements (PPA) (100%, 100%, and 97.1%) and negative percent agreements (NPA) (95.2%, 99.5%, and 99.6%) for influenza A and B viruses and RSV, respectively. In conclusion, this multicenter study using the Cepheid Xpert Flu+RSV Xpress Assay demonstrated high sensitivities and specificities for influenza A and B viruses and RSV in ∼60 min for use at the point-of-care in the CW setting.

Youth living with HIV (YLWH) are at risk for depression. Depressive symptoms can impact treatment engagement, health outcomes, and quality of life. Early identification of symptoms can guide treatment planning. This study aimed to identify trends in depressive symptoms for YLWH in a specialty-care clinic and follow-up clinical treatment procedures. An archival review of a clinical database provided depression screening information for a sample of 130 YLWH between 11 and 25 years old in the southeastern United States. Findings indicated that approximately 24% of the sample screened positive for depression-risk. Most commonly endorsed symptoms included fatigue (54.3%) and sleep difficulties (48.5%). Depressive symptoms did not differ significantly by age, gender, race, ethnicity, or sexual orientation. Youth who acquired HIV behaviorally were more likely to endorse the critical item (i.e., self-harm and/or suicidal ideation) than youth who acquired HIV perinatally. Forty-percent of the sample (i.e., 51 youth) had a follow-up treatment plan. YLWH who endorsed the critical item were more likely to receive follow-up action when compared to those who did not endorse the item. Despite limitations of the study, findings have important implications for clinical care and future research.

We assessed the decisional capacity (DC) of 72 youth with HIV, ages 13–24, using the MacArthur Competence Tool for Treatment, a structured interview that assesses DC along the following dimensions: understanding, appreciation, reasoning, and the ability to express a choice. Using previously established cutoff scores, results suggested 100% of youth were competent in the area of appreciation, but only 62% and 60% were competent in the areas of understanding and reasoning, respectively. Additional descriptive analyses reveal more detailed information regarding specific strengths and weaknesses within each of the dimensions of decisional capacity. These findings have important implications for health literacy initiatives, medical education, and treatment for youth with HIV, and support the need for adherence and secondary prevention interventions that include a decisional capacity component.

The purpose of this study was to assess (a) perceptions of the physician-patient relationship among youth with HIV and (b) the relationship between the perceived physician-patient relationship and disease management variables. Participants were 72 youth with HIV ages 13–24. The Patient Reactions Assessment (PRA) and the Adolescent Patient-Provider Interaction Scale (APPIS) were administered. Results suggest positive perceptions among youth with HIV about their relationship with their physicians. There was a significant correlation between PRA and APPIS scores. However, there were no significant correlations between scores on the PRA and APPIS and demographic or disease management variables.

Despite advances in HIV medicine, adherence presents as a barrier to effective treatment for youth. We designed this study to assess medication knowledge, adherence, and factors affecting adherence in youth with HIV. Participants were 72 youth ages 13 to 24 years with perinatally or behaviorally acquired HIV. Demographic data were collected and a self-report adherence interview was administered. Interviews were audio-recorded to allow for qualitative data analysis. Self-reported adherence varied depending on the framing of questions, with participants reporting greater adherence when asked how many doses they had missed within the past 7 days compared to results from a 7-day recall interview. At least 74% of the sample said they sometimes forgot to take their medication. A taxonomic approach to the qualitative analysis revealed internal and external facilitators and barriers to adherence. Findings suggest a need for education and provider support to include strategies to improve adherence.

Medical decisional capacity refers to the ability to understand, appreciate and make meaningful decisions about one’s health. This is an important construct for children living with HIV whose involvement in their medical care has important implications for disease management. In this study, we assessed the relationship between decisional capacity, developmental stage, intellectual ability, and social-emotional functioning of children with and without HIV infection (n=50). We hypothesized a positive correlation between variables, but did not expect to find a difference in decisional capacity between groups. Results provided partial support for our hypotheses. There was a positive relationship between developmental stage and understanding, which is but one dimension of decisional capacity. Children with HIV infection obtained significantly lower scores on measures of intellectual and adaptive functioning, but there was no significant difference in decisional capacity between groups. Findings suggest that children living with HIV have the capacity to meaningfully participate in their health care despite lower intellectual and adaptive functioning.

OBJECTIVE HIV-1 replication and microbial translocation occur concomitant with systemic immune activation. This study delineates mechanisms of immune activation and CD4 T-cell decline in pediatric HIV-1 infection. DESIGN Cross-sectional and longitudinal cellular and soluble plasma markers for inflammation were evaluated in 14 healthy and 33 perinatally HIV-1-infected pediatric study volunteers prior to and over 96 weeks of protease-inhibitor-containing combination antiretroviral therapy (ART). All HIV-1-infected patients reconstituted CD4 T cells either with suppression of viremia or rebound of drug-resistant virus. METHODS Systemic immune activation was determined by polychromatic flow cytometry of blood lymphocytes and ELISA for plasma soluble CD27, soluble CD14, and tumor necrosis factor. Microbial translocation was evaluated by limulus amebocyte lysate assay to detect bacterial lipopolysaccharide (LPS) and ELISA for antiendotoxin core antigen immunoglobulin M (IgM) antibodies. Immune activation markers were compared with viral load, CD4 cell percentage, and LPS by regression models. Comparisons between healthy and HIV-1-infected or between different viral outcome groups were performed by nonparametric rank sum. RESULTS Microbial translocation was detected in healthy infants but resolved with age (P < 0.05). LPS and soluble CD14 levels were elevated in all HIV-1-infected patients (P < 0.05 and P < 0.0001, respectively) and persisted even if CD4 T cells were fully reconstituted, virus optimally suppressed, and lymphocyte activation resolved by ART. Children with CD4 T-cell reconstitution but viral rebound following ART continued to display high levels of soluble CD27. CONCLUSION Microbial translocation in pediatric HIV-1 infection is associated with persistent monocyte/macrophage activation independent of viral replication or T-cell activation.

The purpose of this chapter is to describe the viral cycle of HIV, the impact of HIV on the immune system, and modes of HIV transmission. The clinical course and manifestations of pediatric HIV, distinguishing differences in course and manifestation between children born with HIV and those who acquire HIV later in adolescence or early adulthood, will be presented. Included in this chapter will be a review of the progression of HIV from the time of infection to the development of symptoms as well as staging for HIV/AIDS. Clinical practice guidelines will be described as well as the latest research on antiretroviral treatment. Finally, information will be presented about HIV vaccine research.

Many HIV-infected children treated with protease inhibitors (PI) reconstitute immunity despite viral breakthrough predicting disease progression. We studied a unique cohort of PI treated children with advanced disease who demonstrated sustained CD4 T cell counts but median post therapy viral load rebounded to >4.0 log(10) copies/ml. Phylogenetic relationships between pre- and post-therapy viruses reveals significant bottlenecks for quasispecies with natural polymorphisms mapping outside of protease active site providing selective advantage for emergence. Among discordant subjects post-therapy viruses fell into two phenotypes; high viral loads (median >5.0 log(10) copies/ml) and attenuated post-therapy replication (median

Secondary bacterial infections often complicate respiratory viral infections, but the mechanisms whereby viruses predispose to bacterial disease are not completely understood. We determined the effects of infection with respiratory syncytial virus (RSV), human parainfluenza virus 3 (HPIV-3), and influenza virus on the abilities of nontypeable Haemophilus influenzae and S treptococcus pneumoniae to adhere to respiratory epithelial cells and how these viruses alter the expression of known receptors for these bacteria. All viruses enhanced bacterial adhesion to primary and immortalized cell lines. RSV and HPIV-3 infection increased the expression of several known receptors for pathogenic bacteria by primary bronchial epithelial cells and A549 cells but not by primary small airway epithelial cells. Influenza virus infection did not alter receptor expression. Paramyxoviruses augmented bacterial adherence to primary bronchial epithelial cells and immortalized cell lines by up-regulating eukaryotic cell receptors for these pathogens, whereas this mechanism was less significant in primary small airway epithelial cells and in influenza virus infections. Respiratory viruses promote bacterial adhesion to respiratory epithelial cells, a process that may increase bacterial colonization and contribute to disease. These studies highlight the distinct responses of different cell types to viral infection and the need to consider this variation when interpreting studies of the interactions between respiratory cells and viral pathogens.

Nontypeable Haemophilus influenzae (NTHI) is an important respiratory pathogen. NTHI initiates infection by adhering to the airway epithelium. Here, we report that NTHI interacts with intracellular adhesion molecule 1 (ICAM-1) expressed by respiratory epithelial cells. A fourfold-higher number of NTHI bacteria adhered to Chinese hamster ovary (CHO) cells transfected with human ICAM-1 (CHO-ICAM-1) than to control CHO cells ( P ≤ 0.005). Blocking cell surface ICAM-1 with specific antibody reduced the adhesion of NTHI to A549 respiratory epithelial cells by 37% ( P = 0.001) and to CHO-ICAM-1 cells by 69% ( P = 0.005). Preincubating the bacteria with recombinant ICAM-1 reduced adhesion by 69% ( P = 0.003). The adherence to CHO-ICAM-1 cells of NTHI strains deficient in the adhesins P5, P2, HMW1/2, and Hap or expressing a truncated lipooligosaccharide was compared to that of parental strains. Only strain 1128f − , which lacks the outer membrane protein (OMP) P5-homologous adhesin (P5 fimbriae), adhered less well than its parental strain. The numbers of NTHI cells adhering to CHO-ICAM-1 cells were reduced by 67% ( P = 0.009) following preincubation with anti-P5 antisera. Furthermore, recombinant ICAM bound to an OMP preparation from strain 1128f + , which expresses P5, but not to that from its P5-deficient mutant, confirming a specific interaction between ICAM-1 and P5 fimbriae. Incubation of respiratory epithelial cells with NTHI increased ICAM-1 expression fourfold ( P = 0.001). Adhesion of NTHI to the respiratory epithelium, therefore, upregulates the expression of its own receptor. Blocking interactions between NTHI P5 fimbriae and ICAM-1 may reduce respiratory colonization by NTHI and limit the frequency and severity of NTHI infection.

BACKGROUND: Vancomycin is often added to therapy for meningitis caused by Streptococcus pneumoniae. Tolerant bacteria without classic resistance that escape killing by multiple antibiotics have been reported sporadically. We determined the prevalence of tolerance to vancomycin in pneumococci and its effect on the outcome of meningitis. METHODS: Archival samples of 215 nasopharyngeal (NP) and 113 meningitis isolates were tested for the killing efficacy of vancomycin. Specific DNA sequence changes in a transporter locus were identified for tolerant isolates. Similar tests were conducted prospectively on 517 NP isolates from healthy children. RESULTS: In archival isolates, tolerance to vancomycin was detected in 3.7% of NP and 10.6% of invasive isolates. Patients with meningitis caused by tolerant isolates had a worse estimated 30-day survival than did patients with meningitis caused by nontolerant isolates (49% vs. 86%; P = .048); 62.5% of tolerant archival NP isolates harbored a specific sequence change for pep27 and vex2 (P = .021). Prospective analysis of 517 carriage isolates indicated that 8.1% were tolerant to vancomycin and that 82.1% of tolerant isolates harbored the specified marker gene sequences (P = .001). CONCLUSIONS: Tolerance to vancomycin exists in the population of pneumococci. Tolerant isolates are associated with meningitis of increased mortality, and these isolates can be tracked by specific marker sequences in 2 genes.

This exploratory study examined the degree to which decisional capacity (DC) is associated with measures of self-reported medication adherence. We hypothesized that youth with higher levels of DC would report greater levels of antiretroviral medication adherence. Seventy-two (72) youth with HIV aged 13–24 participated in this study. Data collection included administration of the MacArthur Competence Tool for Treatment and measures of adherence (i.e., seven-day self-report interview, visual analog scale, and biological indicators). Data were analyzed using descriptive statistics, intercorrelations, and multiple and Poisson regression analyses. Youth with HIV who exhibited greater understanding of their disease were more likely to report fewer missed doses in the last seven days. Findings build upon literature in the areas of DC and health literacy and highlight the potential utility of enhancing HIV disease understanding among youth with HIV.

Pediatric acute-onset neuropsychiatric syndrome (PANS) is a subtype of obsessive compulsive disorder (OCD) marked by an abrupt onset or exacerbation of neuropsychiatric symptoms. We aim to characterize the phenotypic presentation of youth with PANS.Forty-three youth (ages 4-14 years) meeting criteria for PANS were assessed using self-report and clinician-administered measures, medical record reviews, comprehensive clinical evaluation, and laboratory measures.Youth with PANS presented with an early age of OCD onset (mean=7.84 years) and exhibited moderate to severe obsessive compulsive symptoms upon evaluation. All had comorbid anxiety and emotional lability, and scored well below normative means on all quality of life subscales. Youth with elevated streptococcal antibody titers trended toward having higher OCD severity, and presented more frequently with dilated pupils relative to youth without elevated titers. A cluster analysis of core PANS symptoms revealed three distinct symptom clusters that included core characteristic PANS symptoms, streptococcal-related symptoms, and cytokine-driven/physiological symptoms. Youth with PANS who had comorbid tics were more likely to exhibit a decline in school performance, visuomotor impairment, food restriction symptoms, and handwriting deterioration, and they reported lower quality of life relative to youth without tics.The sudden, acute onset of neuropsychiatric symptoms, high frequency of comorbidities (i.e., anxiety, behavioral regression, depression, and suicidality), and poor quality of life capture the PANS subgroup as suddenly and severely impaired youth. Identifying clinical characteristics of youth with PANS will allow clinicians to diagnose and treat this subtype of OCD with a more strategized and effective approach.

Invasive fungal infections are increasing in prevalence because of modern support for immunocompromised patients allowing longer survivial times. The risk of fungal infection has also increased because of the use of broad spectrum of antibiotics in this patient population, and the intensity and duration of immunosuppression. The list of antifungal options has expanded with renewed interest in these infections with high morbidity and mortality. Amphotericin B deoxycholate has been and continues to be the mainstay of antifungal agents. Lipid formulations of amphotericin that improve the serum and the tissue level of amphotericin B while decreasing toxicity are being increasingly used. Imidazoles, including fluconazole and itraconazole, have reduced toxicities in comparison with amphotericin B deoxycholate, but their spectrum activity is limited. Echinocandins, including caspofungin and new investigational agents with unique mechanisms of action, offer great promise as antifungal agents either alone or in a combination agent with amphotericin B. Antifungal strategies and antifungal resistance in relationship to clinical outcomes are also discussed. Copyright © 2001 by W.B. Saunders Company

Background: HIV-1 infection or systemic lupus erythematosus [SLE] disrupt B cell homeostasis, reduce memory B cells, and impair function of IgG and IgM antibodies. Objective: To determine how disturbances in B cell populations producing polyclonal antibodies relate to the IgM repertoire, the IgM transcriptome in health and disease was explored at the complementarity determining region 3 [CDRH3] sequence population level. Methods: 454-deep pyrosequencing in combination with a novel analysis pipeline was applied to define populations of IGHM CDRH3 sequences based on absence or presence of somatic hypermutations [SHM] in peripheral blood B cells. Results: HIV or SLE subjects have reduced biodiversity within their IGHM transcriptome compared to healthy subjects, mainly due to a significant decrease in the number of unique combinations of alleles, although recombination machinery was intact. While major differences between sequences without or with SHM occurred among all groups, IGHD and IGHJ allele use, CDRH3 length distribution, or generation of SHM were similar among study cohorts. Antiretroviral therapy failed to normalize IGHM biodiversity in HIV-infected individuals. All subjects had a low frequency of allelic combinations within the IGHM repertoire similar to known broadly-neutralizing HIV-1 antibodies. Conclusions: Polyclonal expansion would decrease overall IgM biodiversity independent of other mechanisms for development of the B cell repertoire. Applying deep sequencing as a strategy to follow development of the IgM repertoire in health and disease provides a novel molecular assessment of multiple points along the B cell differentiation pathway that is highly sensitive for detecting perturbations within the repertoire at the population level.

Human immunodeficiency virus (HIV) type 1 infection perturbs the T-cell receptor (TCR) Vβ repertoire. The TCR CDR3 length diversity of individual Vβ families was examined within CD45RA and CD45RO CD4 T cells to assess the impact of the virus on clonality throughout CD4 T-cell activation and differentiation. A cross-sectional and longitudinal cohort study of 13 HIV-infected and 8 age-matched healthy children and adolescents examined the Vβ CDR3 length profiles within CD4 T-cell subsets by the use of spectratyping. HIV-infected subjects demonstrated higher numbers of perturbations in CD4 CD45RA T cells (5.8 ± 4.9 Vβ families) than healthy individuals (1.6 ± 1.8 Vβ families) (P= 0.04). Surprisingly, CD4 CD45RO central memory T cells from infected subjects showed no increased perturbations compared to the perturbations for the same cells from healthy subjects (2.9 ± 3.1 and 1.1 ± 1.8 Vβ families, respectively;P= 0.11). CD4 CD45RA TCR perturbations were higher among infected subjects with >25% CD4 cells than healthy subjects (mean number of perturbed Vβfamilies, 6.6 ± 5.4;P= 0.04). No correlations between perturbations in CD4 subsets and pretherapy age or viral load were evident. In contrast to CD8 T cells, HIV induces TCR disruptions within CD45RA but not CD45RO CD4 T cells. Therapy-induced viral suppression resulted in increases in thymic output and the normalization of the diversity of TCR within CD45RA CD4 T cells after 2 months of treatment. Perturbations occur prior to CD4 T-cell attrition and normalize with effective antiretroviral therapy. The impact of HIV on the diversity of TCR within naïve, central memory, and effector memory CD4 T cells is distinctly different from that in CD8 T cells.

Advances in laboratory methods have driven improvements in the management and treatment of HIV infection. The methods to accurately and rapidly diagnose HIV infection in infants and children have been outlined in the previous article. In this review, the laboratory evaluation of infected children is described and methods to monitor progression of disease and response to therapy outlined.

The diagnosis of HIV-1 infection in infants and children continues to present challenges. Currently available virologic assays are sensitive and specific and allow early detection of perinatally acquired HIV infection. Identification soon after birth allows for the rapid initiation of antiretroviral therapy and preservation of the infant's immune system. Serologic diagnostic methods, including HIV-ELISA, Western blot, and immunofluorescence Assay can be used to make the diagnosis of HIV infection in infants older than 18 months of age, children, and adolescents. Recently developed rapid tests allow for testing outside clinical sites, provide results in a short period of time, and allow for prompt initiation of effective prophylaxis in cases of exposure particularly maternal to child transmission. We discuss here the diagnostic management of HIV-exposed infants and HIV-infected children.

Study of the epidemiology of invasive infections caused by encapsulated Haemophilus influenzae has been complicated by the poor sensitivity and specificity of the serologic assays used to identify specific capsular polysaccharides. The population structure of these bacteria is highly clonal, however, and serotype is highly correlated with other genetic characteristics. We sought to determine if alleles of the highly conserved phosphoglucose isomerase ( pgi ) gene correspond to the serotypes of encapsulated H. influenzae strains. pgi alleles of 52 well-characterized encapsulated H. influenzae isolates were amplified by PCR, sequenced, and compared to one another and to additional previously reported H. influenzae pgi alleles. Overall, 83% of the strains possessed pgi alleles associated with the major serotype a, b, e, and f clonotypes that cause the most invasive disease in the United States. Six strains (four type a and two type f) had unusual pgi alleles, which suggested that these strains belonged to less common clonotypes of encapsulated bacteria or were actually nontypeable strains. pgi genotyping may provide a simple and stable surrogate for capsular serotyping. Further studies correlating pgi typing with the expression of capsule are likely to increase our understanding of the epidemiology and pathogenesis of these infections.

Invasive infections caused by non-type b encapsulated Haemophilus influenzae have increased in frequency in the last decade. This change prompted us to characterize the genetic relationships of 48 recently isolated invasive H. influenzae type a (Hia), e (Hie), and f (Hif) strains by comparison of restriction digest patterns (RDPs). Recent Hia isolates exhibited moderate genetic diversity, with the majority segregating into two major clonotypes. Recent Hie and, especially, Hif strains displayed considerably restricted genetic diversity. In particular, all but one Hif strain segregated into a single clonotype, and half of these isolates had identical RDPs. These results are consistent with the hypothesis that the increased incidence of disease due to non-type b encapsulated H. influenzae reflects the emergence of hypervirulent clones, especially in the case of Hif. Alternatively, it is possible that non-type b encapsulated H. influenzae strains have limited overall genetic diversity.

Invasive infections caused by non-type b encapsulated Haemophilus influenzae have increased recently. Because capsule is a major virulence factor, capsulation of 62 recently isolated non-serotype b encapsulated strains was examined. Repeated serotyping confirmed only 69.0% of isolates. The combination of slide agglutination and cap genotyping confirmed 78.9% of type a, 100% of type e, and 86.4% of type f strains. Seven nonencapsulated strains may have lost capsulation through homologous recombination. Three strains that could not be serotyped or genotyped failed to hybridize with any cap probe and are probably nontypeable H. influenzae. Of isolates that retained an intact cap locus, 62.2% had evidence of cap amplification. The epidemiology of non-type b encapsulated H. influenzae infections is complicated by the poor specificity of available serologic reagents and by spontaneous capsule loss. Recently isolated invasive non-type b encapsulated H. influenzae frequently have cap amplification, which may contribute to their virulence.

Background HIV infection decreases thymic output and induces chronic T-cell activation. Objective To examine the reconstitution of naive and activated T cells. Methods Extended immune phenotyping of CD4 + and CD8 + T-cell subsets was combined with T-cell receptor rearrangement excision circle (TREC) levels and measures of T-cell receptor repertoire perturbations in CD8 + T-cell subpopulation to define the global effect of HIV-1 on T-cell dynamics. Evaluations before and after therapy were performed in HIV-infected children and compared with those in healthy individuals. Results Ten HIV-infected children and adolescents with a broad range of pretherapy CD4 + T-cell counts were compared with healthy individuals. Pretherapy late activated CD8 + T cells (CD3 + CD8 + CD45RA + CD27 − CD11a bright cells) were expanded among HIV-infected subjects. Successful antiretroviral therapy increased the proportion of naive T cells (CD3 + CD4 + CD45RA + CD27 + CD28 + and CD3 + CD8 + CD45RA + CD27 + CD11a dim cells), with a significant decrease in late activated CD8 + T cells. The proportion of naive CD4 + and CD8 + T cells significantly predicted log 10 TREC copies/10 6 PBMCs in infected children and healthy control subjects, with a negative correlation in late activated CD8 + T cells and activated CD4 + T cells. Treatment re-established Gaussian distributions and decreased oligoclonal expansion within the Vβ repertoire of CD8 + CD45RA + T cells, but compared with that seen in healthy children, the proportion of late activated CD8 + T cells remained increased. Conclusion HIV infection strikingly shifts the proportion of naive and late activated CD45RA + CD8 + T cells. Homeostasis within this T-cell population reflects TREC levels and the extent of T-cell receptor Vβ perturbations.