Annemarie M. C. van Rossum

Efficacy of highly active antiretroviral therapy in HIV-1 infected children.

Although the reduction in HIV-1-related deaths with highly active antiretroviral therapy (HAART) is similar in adults and children, the extent of the changes in two important surrogate markers HIV-1 RNA levels and CD4+ T cell counts, differs widely. In most paediatric studies virological response rates to HAART are inferior to those in adults. This review provides an overview of the paediatric clinical studies using HAART and seeks to improve the understanding of factors that may contribute to success or failure of HAART in children. An overview of all current articles on paediatric clinical trials using HAART is provided. 23 papers were available. HIV-1 RNA loads and CD4+ T cell counts were used as primary outcome measures. Virological response rates were highly variable, both among the different antiretroviral drugs but also among different studies using the same medication. Four studies in which dosages of the administrated protease inhibitor (PI) were adjusted after pharmacokinetic evaluation had superior virological response rates compared with those in which fixed dosages were used. Immunological response rates were more uniform than virological responses. In almost all studies increases of CD4+ T cell counts are reported independent of the extent of the virological response. Side-effects of HAART were generally mild, transient, and of gastrointestinal origin. Significant percentages of patients with serum lipid abnormalities were reported in three paediatric studies. However, signs of clinical lipodystrophy were not observed. The inferior virological response rates, which have been reported in HIV-1 infected children treated with HAART form a reflection of the challenges that are encountered in the treatment of these children. Difficulties with adherence and with the pharmacokinetics of PIs in children require an intensive, child-adjusted approach. A practical approach to therapy in institutions without tertiary care facilities may be induction therapy with a lopinavir containing regimen (lacking a need for therapeutic drug monitoring), to reduce high viral load levels followed by an easily tolerated maintenance regimen, for example containing abacavir or nevirapine.

Carriage of Mycoplasma pneumoniae in the Upper Respiratory Tract of Symptomatic and Asymptomatic Children: An Observational Study

In order to determine the possible asymptomatic carriage of Mycoplasma pneumoniae in the upper respiratory tracts of children, Emiel Spuesens and colleagues investigate the prevalence of M. pneumoniae in symptomatic and asymptomatic children at a hospital in The Netherlands. Please see later in the article for the Editors Summary

Annual Vaccination against Influenza Virus Hampers Development of Virus-Specific CD8+ T Cell Immunity in Children

ABSTRACT Infection with seasonal influenza A viruses induces immunity to potentially pandemic influenza A viruses of other subtypes (heterosubtypic immunity). We recently demonstrated that vaccination against seasonal influenza prevented the induction of heterosubtypic immunity against influenza A/H5N1 virus induced by infection with seasonal influenza in animal models, which correlated with the absence of virus-specific CD8+ T cell responses. Annual vaccination of all healthy children against influenza has been recommended, but the impact of vaccination on the development of the virus-specific CD8+ T cell immunity in children is currently unknown. Here we compared the virus-specific CD8+ T cell immunity in children vaccinated annually with that in unvaccinated children. In the present study, we compared influenza A virus-specific cellular and humoral responses of unvaccinated healthy control children with those of children with cystic fibrosis (CF) who were vaccinated annually. Similar virus-specific CD4+ T cell and antibody responses were observed, while an age-dependent increase of the virus-specific CD8+ T cell response that was absent in vaccinated CF children was observed in unvaccinated healthy control children. Our results indicate that annual influenza vaccination is effective against seasonal influenza but hampers the development of virus-specific CD8+ T cell responses. The consequences of these findings are discussed in the light of the development of protective immunity to seasonal and future pandemic influenza viruses.

Neonatal Procalcitonin Intervention Study (NeoPInS): Effect of Procalcitonin-guided decision making on Duration of antibiotic Therapy in suspected neonatal early-onset Sepsis: A multi-centre randomized superiority and non-inferiority Intervention Study

BackgroundEarly diagnosis and treatment of the newborn infant with suspected sepsis are essential to prevent severe and life threatening complications. Diagnosis of neonatal sepsis is difficult because of the variable and nonspecific clinical presentation. Therefore, many newborns with nonspecific symptoms are started on antibiotic treatment before the presence of sepsis has been proven. With our recently published single-centre intervention study we were able to show that Procalcitonin determinations allowed to shorten the duration of antibiotic therapy in newborns with suspected early-onset sepsis.Methods/DesignThe study is designed as randomized controlled international multicenter intervention trial on the efficacy and safety of Procalcitonin guided treatment. Term and near-term infants (gestational age ≥ 34 0/7 weeks) with suspected sepsis in the first 3 days of life requiring empiric antibiotic therapy will be included. The duration of antibiotic therapy in the standard group is based on the attending physicians assessment of the likelihood of infection (infection unlikely, possible, probable or proven). In the Procalcitonin group, if infection is considered to be unlikely or possible, antibiotic therapy is discontinued when two consecutive Procalcitonin values are within the normal range. Co-primary outcome measures are the duration of antibiotic therapy (superiority aspect of the trial) and the proportion of infants with a recurrence of infection requiring additional courses of antibiotic therapy and/or death in the first month of life (safety of study intervention, non-inferiority aspect of the trial). The number of infants to be included equals 800 per arm. With these numbers the power of the study to demonstrate superiority for duration of antibiotic therapy as well as non-inferiority regarding safety, i.e. excluding a disadvantage difference larger than 2% for the experimental arm, will both be greater than 80%.DiscussionBenefit of the study is a possible limitation of unnecessary use of antibiotics. The results of our first study suggest that there is a low risk on discontinuing antibiotic treatment too early, resulting in the development of a neonatal infection with its morbidity and mortality.Trial registrationThis trial is registered in the U.S. National Institutes of Healths register, located at http://www.clinicaltrials.gov. (NCT00854932).

Macrolide resistance determination and molecular typing of Mycoplasma pneumoniae by pyrosequencing.

The first choice antibiotics for treatment of Mycoplasma pneumoniae infections are macrolides. Several recent studies, however, have indicated that the prevalence of macrolide (ML)-resistance, which is determined by mutations in the bacterial 23S rRNA, is increasing among M. pneumoniae isolates. Consequently, it is imperative that ML-resistance in M. pneumoniae is rapidly detected to allow appropriate and timely treatment of patients. We therefore set out to determine the utility of pyrosequencing as a convenient technique to assess ML-resistance. In addition, we studied whether pyrosequencing could be useful for molecular typing of M. pneumoniae isolates. To this end, a total of four separate pyrosequencing assays were developed. These assays were designed such as to determine a short genomic sequence from four different sites, i.e. two locations within the 23S rRNA gene, one within the MPN141 (or P1) gene and one within the MPN528a gene. While the 23S rRNA regions were employed to determine ML-resistance, the latter two were used for molecular typing. The pyrosequencing assays were performed on a collection of 108 M. pneumoniae isolates. The ML-resistant isolates within the collection (n=4) were readily identified by pyrosequencing. Moreover, each strain was correctly typed as either a subtype 1 or subtype 2 strain by both the MPN141 and MPN528a pyrosequencing test. Interestingly, two recent isolates from our collection, which were identified as subtype 2 strains by the pyrosequencing assays, were found to carry novel variants of the MPN141 gene, having rearrangements in each of the two repetitive elements (RepMP4 and RepMP2/3) within the gene. In conclusion, pyrosequencing is a convenient technique for ML-resistance determination as well as molecular typing of M. pneumoniae isolates.

Results of 2 Years of Treatment with Protease-Inhibitor–Containing Antiretroviral Therapy in Dutch Children Infected with Human Immunodeficiency Virus Type 1

Clinical, virologic, and immunologic responses to treatment that contained either indinavir or nelfinavir (both regimens included zidovudine and lamivudine) were determined in 32 children infected with human immunodeficiency virus type 1 (HIV-1) who participated for >/= 96 weeks in a prospective, open, uncontrolled multicenter trial. The pharmacokinetics of indinavir and of nelfinavir were determined and showed large interindividual differences. After 96 weeks of therapy, 69% and 50% of the patients had an HIV-1 RNA load that was below the HIV assays detection limits of 500 and 40 copies/mL, respectively. Virologic failure was associated with poor compliance and younger age (independent of baseline virus load and receipt of pretreatment). Relative CD4 cell counts increased significantly in relation to the median of the age-specific reference value, from a median of 44% at baseline to 94% after 96 weeks. In a high percentage of the children, clinical, virologic, and immunologic response rates to combination therapy were optimal during the initial 2 years of therapy.

Sequence variations in RepMP2/3 and RepMP4 elements reveal intragenomic homologous DNA recombination events in Mycoplasma pneumoniae

The gene encoding major adhesin protein P1 of Mycoplasma pneumoniae, MPN141, contains two DNA sequence stretches, designated RepMP2/3 and RepMP4, which display variation among strains. This variation allows strains to be differentiated into two major P1 genotypes (1 and 2) and several variants. Interestingly, multiple versions of the RepMP2/3 and RepMP4 elements exist at other sites within the bacterial genome. Because these versions are closely related in sequence, but not identical, it has been hypothesized that they have the capacity to recombine with their counterparts within MPN141, and thereby serve as a source of sequence variation of the P1 protein. In order to determine the variation within the RepMP2/3 and RepMP4 elements, both within the bacterial genome and among strains, we analysed the DNA sequences of all RepMP2/3 and RepMP4 elements within the genomes of 23 M. pneumoniae strains. Our data demonstrate that: (i) recombination is likely to have occurred between two RepMP2/3 elements in four of the strains, and (ii) all previously described P1 genotypes can be explained by inter-RepMP recombination events. Moreover, the difference between the two major P1 genotypes was reflected in all RepMP elements, such that subtype 1 and 2 strains can be differentiated on the basis of sequence variation in each RepMP element. This implies that subtype 1 and subtype 2 strains represent evolutionarily diverged strain lineages. Finally, a classification scheme is proposed in which the P1 genotype of M. pneumoniae isolates can be described in a sequence-based, universal fashion.

Macrolide Resistance Determination and Molecular Typing of Mycoplasma pneumoniae in Respiratory Specimens Collected between 1997 and 2008 in The Netherlands

ABSTRACT An important role in the treatment regimens for Mycoplasma pneumoniae infections is played by macrolide (ML) antibiotics. In the past few years, however, a steady increase has been detected in the worldwide prevalence of ML-resistant (MLr) M. pneumoniae strains. It is obvious that this increase necessitates a continuous monitoring of MLr and, when detected, modification of antibiotic treatment modalities. Previously, we developed a pyrosequencing-based assay system for the genetic determination of MLr as well as molecular typing of M. pneumoniae. In this study, the sensitivity of this system was improved by the inclusion of a nested-PCR protocol. The modified system was applied to 114 M. pneumoniae-positive specimens that were obtained from a collection of 4,390 samples from patients with acute respiratory tract infections. These samples were collected between 1997 and 2008 in The Netherlands. The pyrosequencing system produced reliable data in 86% of the specimens that contained >500 M. pneumoniae genome copies/ml of patient sample. Each of these samples contained DNA of the ML-sensitive genotype. While 43% of the samples were found to harbor the M. pneumoniae subtype 1 genotype, 57% contained the subtype 2 genotype. We conclude that the pyrosequencing-based assay system is a useful tool for MLr determination and molecular typing of M. pneumoniae in patient samples. MLr-associated M. pneumoniae genotypes, however, were not found in the current study population.

Common variable immunodeficiency and idiopathic primary hypogammaglobulinemia: two different conditions within the same disease spectrum

Patients with hypogammaglobulinemia who do not fulfill all the classical diagnostic criteria for common variable immunodeficiency (reduction of two immunoglobulin isotypes and a reduced response to vaccination) constitute a diagnostic and therapeutic dilemma, because information concerning the clinical and immunological characteristics of these patients with idiopathic primary hypogammaglobulinemia is not available. In 44 common variable immunodeficiency and 21 idiopathic primary hypogammaglobulinemia patients we determined the clinical phenotypes and performed flow cytometric immunophenotyping to assess the pathophysiological B-cell patterns and memory B-cell subset counts. Age-matched B-cell subset reference values of 130 healthy donors were generated. Severe pneumonia and bronchiectasis occurred at similar frequencies in idiopathic primary hypogammaglobulinemia and common variable immunodeficiency. Although IgG levels were only moderately reduced compared to common variable immunodeficiency, 12 of 21 idiopathic primary hypogammaglobulinemia patients required immunoglobulin replacement. Non-infectious disease-related clinical phenotypes (autoimmune cytopenia, polyclonal lymphocytic proliferation and persistent unexplained enteropathy) were exclusively observed in common variable immunodeficiency and were associated with early peripheral B-cell maturation defects or B-cell survival defects. T-cell dependent memory B-cell formation was more severely affected in common variable immunodeficiency. Furthermore, 14 of 21 idiopathic primary hypogammaglobulinemia patients showed normal peripheral B-cell subset counts, suggestive for a plasma cell defect. In conclusion, idiopathic primary hypogammaglobulinemia patients who do not fulfill all diagnostic criteria of common variable immunodeficiency have moderately decreased immunoglobulin levels and often a normal peripheral B-cell subset distribution, but still suffer from serious infectious complications.

Sustained viral suppression and immune recovery in HIV type 1-infected children after 4 years of highly active antiretroviral therapy

We report the data from a long-term study of 31 human immunodeficiency virus type 1 (HIV-1)-infected children who were treated with highly active antiretroviral therapy. A high proportion of the children had undetectable HIV-1 RNA levels. CD4+ T cell counts recovered and remained stable. Adverse events were observed frequently but were mostly mild.