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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.

BACKGROUND: Mycoplasma pneumoniae is thought to be a common cause of respiratory tract infections (RTIs) in children. The diagnosis of M. pneumoniae RTIs currently relies on serological methods and/or the detection of bacterial DNA in the upper respiratory tract (URT). It is conceivable, however, that these diagnostic methods also yield positive results if M. pneumoniae is carried asymptomatically in the URT. Positive results from these tests may therefore not always be indicative of a symptomatic infection. The existence of asymptomatic carriage of M. pneumoniae has not been established. We hypothesized that asymptomatic carriage in children exists and investigated whether colonization and symptomatic infection could be differentiated by current diagnostic methods. METHODS AND FINDINGS: This study was conducted at the Erasmus MC-Sophia Children's Hospital and the after-hours General Practitioners Cooperative in Rotterdam, The Netherlands. Asymptomatic children (n = 405) and children with RTI symptoms (n = 321) aged 3 mo to 16 y were enrolled in a cross-sectional study from July 1, 2008, to November 30, 2011. Clinical data, pharyngeal and nasopharyngeal specimens, and serum samples were collected. The primary objective was to differentiate between colonization and symptomatic infection with M. pneumoniae by current diagnostic methods, especially real-time PCR. M. pneumoniae DNA was detected in 21.2% (95% CI 17.2%-25.2%) of the asymptomatic children and in 16.2% (95% CI 12.2%-20.2%) of the symptomatic children (p = 0.11). Neither serology nor quantitative PCR nor culture differentiated asymptomatic carriage from infection. A total of 202 children were tested for the presence of other bacterial and viral pathogens. Two or more pathogens were found in 56% (63/112) of the asymptomatic children and in 55.5% (50/90) of the symptomatic children. Finally, longitudinal sampling showed persistence of M. pneumoniae in the URT for up to 4 mo. Fifteen of the 21 asymptomatic children with M. pneumoniae and 19 of the 22 symptomatic children with M. pneumoniae in this longitudinal follow-up tested negative after 1 mo. CONCLUSIONS: Although our study has limitations, such as a single study site and limited sample size, our data indicate that the presence of M. pneumoniae in the URT is common in asymptomatic children. The current diagnostic tests for M. pneumoniae are unable to differentiate between asymptomatic carriage and symptomatic infection.

Objectives: Zidovudine is often administered every 12?h in HIV-infected children, but so far no pharmacokinetic data are available for the administration of this agent every 12?h. We have evaluated the plasma pharmacokinetics of zidovudine administered every 8?h versus every 12?h in HIV-1-infected children.Methods: In HIV-1-infected children who switched from zidovudine every 8?h to every 12?h, a pharmacokinetic curve was recorded both before and after the switch. Zidovudine plasma levels were measured by HPLC. Pharmacokinetic parameters were calculated by non-compartmental methods.Results: Six HIV-1-infected children [median age (range) 7.8 (2.513.4) years] were included. In these patients, geometric mean ratios of AUC024 and Cmax for zidovudine every 12?h versus every 8?h were not significantly different from 1.0.Conclusions: The plasma pharmacokinetic parameters of zidovudine taken every 8?h and every 12?h were not significantly different and therefore suggest bioequivalence of these two dose frequencies. [ABSTRACT FROM AUTHOR]

Introduction High morbidity and mortality rates of proven bacterial infection are the main reason for substantial use of intravenous antibiotics in neonates during the first week of life. In older children, intravenous-to-oral switch after 48 hours of intravenous therapy has been shown to have many advantages and is nowadays commonly practised. We, therefore, aim to evaluate the effectiveness, safety and cost-effectiveness of an early intravenous-to-oral switch in neonates with a probable bacterial infection.Methods and analysis We present a protocol for a multicentre randomised controlled trial assessing the non-inferiority of an early intravenous-to-oral antibiotic switch compared with a full course of intravenous antibiotics in neonates (0–28 days of age) with a probable bacterial infection. Five hundred and fifty patients will be recruited in 17 hospitals in the Netherlands. After 48 hours of intravenous treatment, they will be assigned to either continue with intravenous therapy for another 5 days (control) or switch to amoxicillin/clavulanic acid suspension (intervention). Both groups will be treated for a total of 7 days. The primary outcome will be bacterial (re)infection within 28 days after treatment completion. Secondary outcomes are the pharmacokinetic profile of oral amoxicillin/clavulanic acid, the impact on quality of life, cost-effectiveness, impact on microbiome development and additional yield of molecular techniques in diagnosis of probable bacterial infection.Ethics and dissemination This study has been approved by the Medical Ethics Committee of the Erasmus Medical Centre. Results will be presented in peer-reviewed journals and at international conferences.Trial registration number NCT03247920

The co-inhibitory immune receptor carcinoembryonic antigen-related cell-adhesion molecule 1 (CEACAM1) and its self-ligand CEACAM1 can suppress T cell function. Suppression of T cell function in sepsis is well documented. Late-onset neonatal sepsis in VLBW-infants was associated with an increased percentage CEACAM1 positive CD4(+) T-cells. Meningococcal septic shock in children was associated with increased serum soluble CEACAM1. In conclusion our data demonstrate increased surface expression of the co-inhibitory immune receptor CEACAM1 in late-onset neonatal sepsis in VLBW-infants, and increased circulating soluble CEACAM1 in children with meningococcal sepsis. Increased T-cell CEACAM1 expression and increased circulating soluble CEACAM1 may contribute to sepsis-associated immune suppression.

The DNA recombination and repair machinery of Mycoplasma pneumoniae is composed of a limited set of approximately 11 proteins. Two of these proteins were predicted to be encoded by neighboring open reading frames (ORFs) MPN340 and MPN341. Both ORFs were found to have sequence similarity with genes that encode proteins belonging to the DNA helicase superfamily 1 (SF1). Interestingly, while a homolog of the MPN341 ORF is present in the genome of Mycoplasma genitalium (ORF MG244), MPN340 is an M. pneumoniae-specific ORF that is not found in other mycoplasmas. Moreover, the length of MPN340 (1590 base pairs [bp]) is considerably shorter than that of MPN341 (2148 bp). Examination of the MPN340-encoded amino acid sequence indicated that it may lack a so-called 2B subdomain, which is found in most SF1 DNA helicases. Also, the MPN340-encoded amino acid sequence was found to differ between subtype 1 strain M129 and subtype 2 strain FH at three amino acid positions. Both protein variants, which were termed PcrA(s) M129 and PcrA(s) FH, respectively, as well as the MPN341- and MG244-encoded proteins (PcrA Mpn and PcrA Mge , respectively), were purified, and tested for their ability to interact with DNA. While PcrA Mpn and PcrA Mge were found to bind preferentially to single-stranded DNA, both PcrA(s) M129 and PcrA(s) FH did not demonstrate significant DNA binding. However, all four proteins were found to have divalent cation- and ATP-dependent DNA helicase activity. The proteins displayed highest activity on partially double-stranded DNA substrates carrying 3' single-stranded extensions.

The DNA recombination and repair machineries of Mycoplasma genitalium and Mycoplasma pneumoniae differ considerably from those of gram-positive and gram-negative bacteria. Most notably, M. pneumoniae is unable to express a functional RecU Holliday junction (HJ) resolvase. In addition, the RuvB homologues from both M. pneumoniae and M. genitalium only exhibit DNA helicase activity but not HJ branch migration activity in vitro. To identify a putative role of the RuvA homologues of these mycoplasmas in DNA recombination, both proteins (RuvA(Mpn) and RuvA(Mge), respectively) were studied for their ability to bind DNA and to interact with RuvB and RecU. In spite of a high level of sequence conservation between RuvA(Mpn) and RuvA(Mge) (68.8% identity), substantial differences were found between these proteins in their activities. First, RuvA(Mge) was found to preferentially bind to HJs, whereas RuvA(Mpn) displayed similar affinities for both HJs and single-stranded DNA. Second, while RuvA(Mpn) is able to form two distinct complexes with HJs, RuvA(Mge) only produced a single HJ complex. Third, RuvA(Mge) stimulated the DNA helicase and ATPase activities of RuvB(Mge), whereas RuvA(Mpn) did not augment RuvB activity. Finally, while both RuvA(Mge) and RecU(Mge) efficiently bind to HJs, they did not compete with each other for HJ binding, but formed stable complexes with HJs over a wide protein concentration range. This interaction, however, resulted in inhibition of the HJ resolution activity of RecU(Mge).

HIV protease inhibitors must penetrate into cells to exert their action. Differences in the intracellular pharmacokinetics of these drugs may explain why some patients fail on therapy or suffer from drug toxicity. Yet, there is no information available on the intracellular levels of HIV protease inhibitors in HIV infected children, which is in part due to the large amount of sample that is normally required to measure the intracellular concentrations of these drugs. Therefore, we developed an ultra-fast and sensitive assay to measure the intracellular concentrations of HIV protease inhibitors in small amounts of peripheral blood mononuclear cells (PBMCs), and determined the intracellular concentrations of lopinavir and ritonavir in HIV infected children. An assay based on matrix-assisted laser desorption/ionization (MALDI)-triple quadrupole mass spectrometry was developed to determine the concentrations of HIV protease inhibitors in 10 microL plasma and 1x10(6) PBMCs. Precisions and accuracies were within the values set by the FDA for bioanalytical method validation. Lopinavir and ritonavir did not accumulate in PBMCs of HIV infected children. In addition, the intracellular concentrations of lopinavir and ritonavir correlated poorly to the plasma concentrations of these drugs. MALDI-triple quadrupole mass spectrometry is a new tool for ultra-fast and sensitive determination of drug concentrations which can be used, for example, to assess the intracellular pharmacokinetics of HIV protease inhibitors in HIV infected children.