Carina A. Rodriguez

Respiratory Viruses Augment the Adhesion of Bacterial Pathogens to Respiratory Epithelium in a Viral Species- and Cell Type-Dependent Manner

ABSTRACT 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 Streptococcus 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.

Microbial translocation induces persistent macrophage activation unrelated to HIV-1 levels or T cell activation following therapy

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.

Nontypeable Haemophilus influenzae Adheres to Intercellular Adhesion Molecule 1 (ICAM-1) on Respiratory Epithelial Cells and Upregulates ICAM-1 Expression

ABSTRACT 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.

Prevalence and distribution of adhesins in invasive non-type b encapsulated Haemophilus influenzae

ABSTRACT Adhesion to the respiratory epithelium plays an important role in Haemophilus influenzae infection. The distribution of H. influenzae adhesins in type b and nontypeable strains has been characterized, but little is known about the prevalence of these factors in non-type b encapsulated strains. We analyzed 53 invasive type a, type e, and type f strains for the presence of hap, hia, hmw, and hif genes; Hap, Hia, and HMW1/2 adhesins; and hemagglutinating pili. The hap gene was ubiquitous, and homologs of hmw and hia were present in 7 of 53 (13.2%) and 45 of 53 (84.9%) strains, respectively. Hap was detected in 28 of 45 (62.2%) hap+ strains, HMW1/2 was detected in 5 of 7 (71.4%) hmw+ strains, and Hia was detected in 31 of 45 (68.8%) hia+ strains. The hif gene cluster was present in 26 of 53 strains (49.1%), and 21 of 26 hif+ strains (80.8%) agglutinated (HA) red blood cells. Nine isolates exhibited HA but lacked the hif gene cluster. The distribution of adhesin genes correlated with the genetic relatedness of the strains. Strains belonging to one type a clonotype and the major type e clonotype possessed hia but lacked the hif cluster. Strains belonging to the second type a clonotype possessed both hia and hif genes. All type f strains belonging to the major type f clonotype possessed hia and lacked hifB. Although the specific complement of adhesin genes in non-type b encapsulated H. influenzae varies, most invasive strains express Hap and Hia, suggesting these adhesins may be especially important to the virulence of these organisms.

Limited Genetic Diversity of Recent Invasive Isolates of Non-Serotype b Encapsulated Haemophilus influenzae

ABSTRACT 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.

Tolerance to Vancomycin in Pneumococci: Detection with a Molecular Marker and Assessment of Clinical Impact

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.

Capsulation Loci of Non-Serotype b Encapsulated Haemophilus influenzae

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.

Characterization of the Pediatric Acute-Onset Neuropsychiatric Syndrome Phenotype

OBJECTIVE 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. METHODS 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. RESULTS 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. CONCLUSIONS 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.

Antiretroviral Therapy Restores Diversity in the T-Cell Receptor Vβ Repertoire of CD4 T-Cell Subpopulations among Human Immunodeficiency Virus Type 1-Infected Children and Adolescents

ABSTRACT 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 Vβ families, 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.

Psychiatric symptoms and antiretroviral nonadherence in US youth with perinatal HIV: a longitudinal study.

Objectives:The relationship of specific psychiatric conditions to adherence has not been examined in longitudinal studies of youth with perinatal HIV infection (PHIV). We examined associations between psychiatric conditions and antiretroviral nonadherence over 2 years. Design:Longitudinal study in 294 PHIV youth, 6–17 years old, in the United States and Puerto Rico. Methods:We annually assessed three nonadherence outcomes: missed above 5% of doses in the past 3 days, missed a dose within the past month, and unsuppressed viral load (>400 copies/ml). We fit multivariable logistic models for nonadherence using Generalized Estimating Equations, and evaluated associations of psychiatric conditions (attention deficit hyperactivity disorder, disruptive behavior, depression, anxiety) at entry with incident nonadherence using multivariable logistic regression. Results:Nonadherence prevalence at study entry was 14% (3-day recall), 32% (past month nonadherence), and 38% (unsuppressed viral load), remaining similar over time. At entry, 38% met symptom cut-off criteria for at least one psychiatric condition. Greater odds of 3-day recall nonadherence were observed at week 96 for those with depression [adjusted odds ratio (aOR) 4.14, 95% confidence interval (CI) 1.11–15.42] or disruptive behavior (aOR 3.36, 95% CI 1.02–11.10], but not at entry. Those with vs. without attention deficit hyperactivity disorder had elevated odds of unsuppressed viral load at weeks 48 (aOR 2.46, 95% CI 1.27–4.78) and 96 (aOR 2.35, 95% CI 1.01–5.45), but not at entry. Among 232 youth adherent at entry, 16% reported incident 3-day recall nonadherence. Disruptive behavior conditions at entry were associated with incident 3-day recall nonadherence (aOR 3.01, 95% CI 1.24–7.31). Conclusion:In PHIV youth, comprehensive adherence interventions that address psychiatric conditions throughout the transition to adult care are needed.