Tom F. W. Wolfs

HIV-1 genomic rna diversification following sexual and parenteral virus transmission☆

Human immunodeficiency virus type 1 (HIV-1) genomic RNA variation was studied in seven presumed donor-recipient pairs directly following sexual (6/7) or parenteral (1/7) transmission. The first RNA-positive serum sample of each recipient and the serum sample of the virus transmitter, identified by epidemiological history and taken within a time bracket of three months of the recipient seroconversion, were analyzed by polymerase chain reaction amplification followed by sequencing of eight cDNA clones of 276 bp, including the V3 coding region. The sequence populations of the recipients were without exception homogeneous, while the sequence populations of the transmitters showed varying degrees of heterogeneity. Nucleotide distance between consensus sequences of unrelated individuals from the Amsterdam population (interpatient variation) averaged 11% (range 7-15%). The largest distance between two clonal sequences of one individual (intrapatient variation) was also 11%. Consensus sequences of five recipients differed by only 0-1% from the consensus sequence of the presumed transmitter, including two pairs of which the transmission was either proven or highly probable. This contrasted with a difference of 10-12% in two pairs, casting doubt on the epidemiological relatedness. Antibody reactivity to a panel of V3 peptides with varying degrees of similarity to the V3 sequences obtained did not augment the discriminatory power of sequence analysis. Results of the sequential sequencing of samples of one transmitter suggest that this was due to an anamnestic antibody response of the transmitter to early variants. From the loss of sequence heterogeneity following transmission and the consensus sequence similarities observed within five transmitter-recipient pairs, we conclude that HIV-1 transmission results in the selection of a limited number of genomes carrying on the infection in the new host, but does not generally lead to a shift in the sequence population as defined by the consensus sequence.

Naturally occurring mutations within HIV-1 V3 genomic RNA lead to antigenic variation dependent on a single amino acid substitution

In a study on the evolution of genomic diversity of HIV-1, genomic RNA was isolated from serum of two individuals. Starting at the time of primary infection we collected six samples of serum from each patient over a period of 5 years. Ninety-four cDNA clones (50 of patient 1 and 44 of patient 495) of part of the envelope coding region including the principal neutralization domain (PND) were sequenced. Around the time of antibody seroconversion, genomic RNA levels reached a peak and the population of sequences was highly homogeneous. In the course of the infection, the number of amino acid substitutions accumulated, which led to a higher genomic diversity within successive samples and a drift in the consensus sequence, progressively differing from the first found consensus sequence. Fixation of a substitution at glycoprotein 120 amino acid 308 was observed in both patients between two time points (patient 1, H----P; patient 495, P----H). With the use of 16-meric synthetic peptides, differing only at the 308 position (H308 versus P308), antibody binding specificity was found to be dependent on this difference. In patient 495, the nonconservative (P308----H) substitution reduced the binding affinity with the patients antibodies. Furthermore, antibody competition assays showed that the observed substitution at position 308 elicited a new antibody population, indicating antigenic variation. After the decline of V3-specific antibodies, the simultaneous increase in genomic RNA levels and progression to AIDS in patient 495, a new variant with major changes in the PND emerged, again forming a homogeneous population of sequences.

Maintenance azithromycin treatment in pediatric patients with cystic fibrosis: long-term outcomes related to macrolide resistance and pulmonary function.

Background: Maintenance azithromycin therapy may improve pulmonary function in patients with cystic fibrosis (CF) with Pseudomonas aeruginosa infection because of its antiinflammatory properties. However, azithromycin therapy might increase macrolide resistance in Staphylococcus aureus cultured from respiratory secretions. We studied the emergence of macrolide resistance in S. aureus and correlated this to pulmonary function decline in pediatric patients with CF on daily azithromycin therapy. Methods: Respiratory cultures of 100 patients with CF were analyzed for S. aureus colonization and its resistance pattern before and during 3 years after initiation of azithromycin maintenance therapy. Mean annual change in forced expiratory volume as percent of predicted (FEV1 %) was calculated to compare pulmonary function before and after azithromycin therapy. Results: Staphylococcal colonization did not significantly decrease after initiation of azithromycin (50% versus 48%). Before start of therapy, 10% of patients with staphylococcal colonization had macrolide-resistant strains. Staphylococcal resistance increased to 83% in the first year; 97% in the second and 100% in the third year after initiation of azithromycin therapy (P < 0.001). Half of macrolide-resistant S. aureus comprised the macrolide–lincosamide–streptogramin phenotype. Percent forced expiratory volume in 1 second improved in the first year after initiation of azithromycin (mean annual change: −4.75% before versus +3.09% after initiation; P < 0.01) but decreased during the second and third years after initiation (−5.15% and −3.65%, respectively). Emergence of macrolide-resistant S. aureus was not related to pulmonary function decline. Conclusion: Maintenance azithromycin therapy in patients with CF leads to macrolide resistance in nearly all S. aureus carriers. Pulmonary function improvement after initiation of azithromycin therapy seems to be temporary and appears not to be related to macrolide resistance of S. aureus.

Increased Detection of Respiratory Syncytial Virus, Influenza Viruses, Parainfluenza Viruses, and Adenoviruses with Real-Time PCR in Samples from Patients with Respiratory Symptoms

ABSTRACT Respiratory samples (n = 267) from hospitalized patients with respiratory symptoms were tested by real-time PCR, viral culture, and direct immunofluorescence for respiratory syncytial virus, influenza virus, parainfluenza viruses, and adenoviruses. Compared with conventional diagnostic tests, real-time PCR increased the diagnostic yields for these viruses from 24% to 43% and from 3.5% to 36% for children and adults, respectively.

Respiratory Pathogens in Children with and without Respiratory Symptoms

Objectives To investigate the occurrence of respiratory pathogens in samples from children with and without respiratory symptoms and to identify whether age and/ or coinfections modify the impact of respiratory pathogens on symptoms. Study design In a prospective longitudinal study, 18 children were sampled biweekly for respiratory pathogens, irrespective of respiratory symptoms. Polymerase chain reaction was performed for 13 respiratory pathogens. Episodes were defined “asymptomatic” if no symptoms of any respiratory tract illness were present between 1 week before and 1 week after sampling. Results A total of 230 samples were collected. In 56% of the symptomatic episodes, a pathogen was detected, compared with 40% of the asymptomatic episodes (P = .03). Rhinovirus and coronaviruses were most prevalent in both symptomatic and asymptomatic episodes. In the youngest children, 9% of the pathogen-positive episodes were asymptomatic, compared with 36% in the oldest children (P = .01). Multiple pathogens were found in 17% of the symptomatic episodes and in 3% of the asymptomatic episodes (P = .02). Conclusions Respiratory pathogens are frequently detected in samples from children with no respiratory symptoms. Symptomatic cases occurred more often in younger children and with detections of more than 1 respiratory pathogen.

A Systemic Neutrophil Response Precedes Robust CD8+ T-Cell Activation during Natural Respiratory Syncytial Virus Infection in Infants

ABSTRACT Severe primary respiratory syncytial virus (RSV) infections are characterized by bronchiolitis accompanied by wheezing. Controversy exists as to whether infants suffer from virus-induced lung pathology or from excessive immune responses. Furthermore, detailed knowledge about the development of primary T-cell responses to viral infections in infants is lacking. We studied the dynamics of innate neutrophil and adaptive T-cell responses in peripheral blood in relation to theviral load and parameters of disease in infants admitted to the intensive care unit with severe RSV infection. Analysis of primary T-cell responses showed substantial CD8+ T-cell activation, which peaked during convalescence. A strong neutrophil response, characterized by mobilization of bone marrow-derived neutrophil precursors, preceded the peak in T-cell activation. The kinetics of this neutrophil response followed the peak of clinical symptoms and the viral load with a 2- to 3-day delay. From the sequence of events, we conclude that CD8+ T-cell responses, initiated during primary RSV infections, are unlikely to contribute to disease when it is most severe. The mobilization of precursor neutrophils might reflect the strong neutrophil influx into the airways, which is a characteristic feature during RSV infections and might be an integral pathogenic process in the disease.

RSV Mediates Pseudomonas aeruginosa Binding to Cystic Fibrosis and Normal Epithelial Cells

Cystic fibrosis lung disease typically has a course of exacerbations and remissions, suggesting that external factors like viral infections can influence this course. Clinical data suggest synergism between respiratory syncytial virus (RSV) infections and Pseudomonas aeruginosa in cystic fibrosis (CF) lung disease. We studied the influence of RSV infection on adherence of P. aeruginosa to IB3-1, HEp-2, and A549 epithelial cell monolayers in vitro. RSV infection of epithelial cells as well as simultaneous addition of RSV and P. aeruginosa to noninfected cells both strongly enhanced the pseudomonal adherence to epithelial cells. The increased adherence varied from 1.2- to 8.2-fold in case of previous RSV infection, and from 1.7- to 16.1-fold in case of simultaneous addition. We observed direct binding of RSV to P. aeruginosa, and blocking of RSV with heparin eliminated the effect on increased adherence. This suggests that RSV possibly acts as a coupling agent between P. aeruginosa and epithelial cells. In conclusion, RSV enhances P. aeruginosa infection of respiratory epithelial cells. It suggests a role of specific viral–bacterial interactions in exacerbations of CF lung disease, which could have important implications on prevention and treatment.

Prevalence and impact of respiratory viral infections in young children with cystic fibrosis: prospective cohort study.

OBJECTIVE. We aimed to investigate differences in upper and lower respiratory tract symptoms in relation to respiratory viral infections detected with polymerase chain reaction assays in young children with cystic fibrosis and healthy control subjects. METHODS. In a 6-month winter period, 20 young children with cystic fibrosis and 18 age-matched, healthy, control subjects were contacted twice per week for detection of symptoms of an acute respiratory illness. If any symptom was present, then a home visit was made for physical examination and collection of nasopharyngeal swabs for viral analysis. In addition, parents were instructed to collect nasopharyngeal swabs every 2 weeks. RESULTS. Children with cystic fibrosis and healthy control subjects had similar frequencies of acute respiratory illnesses (3.8 ± 1.0 and 4.2 ± 1.7 episodes, respectively). Although there were no significant differences in upper respiratory tract symptoms, the children with cystic fibrosis had longer periods of lower respiratory tract symptoms (22.4 ± 22.2 vs 12.8 ± 13.8 days) and a higher mean severity score per episode (2.35 ± 0.64 vs 1.92 ± 0.46). In addition, similar increases in upper respiratory tract symptom scores were associated with significantly greater increases in lower respiratory tract symptom scores in children with cystic fibrosis. No differences in the seasonal occurrences and distributions of respiratory viruses were observed, with picornaviruses and coronaviruses being the most prevalent. CONCLUSIONS. Although there were no differences in the seasonal occurrences and distributions of polymerase chain reaction-detected respiratory viruses, acute respiratory illnesses were frequently associated with increased lower respiratory tract morbidity in young children with cystic fibrosis.

Neonatal Sepsis by Campylobacter jejuni: Genetically Proven Transmission from a Household Puppy

We report a case of neonatal Campylobacter jejuni sepsis in a 3-week-old infant who acquired the infection through transmission from a recently acquired household puppy. Genotyping of Campylobacter strains obtained from puppy and child resulted in highly homogeneous findings. This represents the first genetically proven C. jejuni dog-human transmission.

Loss of antibodies and response to (re-)vaccination in children after treatment for acute lymphocytic leukemia : a systematic review

Intensified chemotherapy regimens resulting in improved survival of children with acute lymphocytic leukemia (ALL) lead to concerns about therapy-induced immune damage reflected by the loss of protection of previous immunizations and the efficacy of (re-)vaccination. The severity of secondary immunodeficiency, however, is not clear and knowledge is based on a limited number of studies. We performed a systematic review on literature concerning vaccination data of children with ALL published since 1980. Eight studies fulfilled the inclusion criteria. Regarding antibody titers after treatment, the number of children who had preserved the defined protection level for antibodies differed widely, ranging from 17 to 98% for diphtheria, 27 to 82% for Bordetella pertussis, 20 to 98% for tetanus, 62 to 100% for poliomyelitis, 35 to 100% for Haemophilus influenzae type B (HiB), 29 to 92% for mumps, 29 to 60% for measles and 72 to 92% for rubella. Most patients however responded to revaccination, demonstrating immunological recovery. Although the designs and results of the included studies varied widely, it can be concluded that cytostatic therapy for ALL in children results in a temporarily reduction of specific antibody levels. Memory is preserved but revaccination may be warranted. This is the first systematic review and the best possible current approximation of chemotherapy-induced immune damage in children after ALL treatment.