Ian C. Michelow

The mannose-binding lectin: a prototypic pattern recognition molecule.

The innate immune system is comprised of a sophisticated network of recognition and effector molecules that act together to protect the host in the first minutes or hours of exposure to an infectious challenge. The mannose-binding lectin (MBL) is an evolutionary conserved circulating host defense protein that acts as a broad-spectrum recognition molecule against a wide variety of infectious agents. Target binding triggers the MBL pathway of complement activation. MBL can be considered conceptually as an ‘ante-antibody’ because it has a role in mammals during the lag period that is required to develop an antibody response against infectious agents. Additionally, there are MBL-like homologues in animals that lack adaptive immunity that activate a primitive complement system, and under these circumstances these MBL-like molecules play an analogous role to antibodies in higher animals. These molecules might be considered to be functional antecedents of antibodies. Recent work also indicates that MBL recognizes altered self-antigens, and as such MBL has a role that extends beyond a traditional role in first line host defense as it appears to play a role as a modulator of inflammation.

Diagnosis of Streptococcus pneumoniae Lower Respiratory Infection in Hospitalized Children by Culture, Polymerase Chain Reaction, Serological Testing, and Urinary Antigen Detection

A prospective study of 154 consecutive high-risk hospitalized children with lower respiratory infections was conducted to determine the clinical utility of a pneumolysin-based polymerase chain reaction (PCR) assay compared with blood and pleural fluid cultures and serological and urinary antigen tests to determine the incidence of Streptococcus pneumoniae. Whole blood, buffy coat, or plasma samples from 67 children (44%) tested positive by PCR. Sensitivity was 100% among 11 promptly tested culture-confirmed children and specificity was 95% among control subjects. Age, prior oral antibiotic therapy, and pneumococcal nasopharyngeal colonization did not influence PCR results, whereas several surrogates of disease severity were associated with positive tests. Although serological and urinary antigen tests had comparable sensitivity, specificity varied among infected children, and statistical agreement among all assays was limited. These findings support the use of PCR tests to evaluate the protective efficacy of pneumococcal conjugate vaccines and to identify promptly children with pretreated or nonbacteremic pneumococcal lower respiratory infections.

Fecal Bacteriotherapy for Relapsing Clostridium difficile Infection in a Child: A Proposed Treatment Protocol

Clostridium difficile infection (CDI) is a potentially serious emerging infectious disease. The incidences of CDI in childhood and CDI cases complicated by relapses have increased by 50% or more in North America during the past 2 decades. We report here the case of a 2-year-old child with relapsing CDI caused by the epidemic strain BI/NAP1/O27 that was refractory to Saccharomyces boulardii and Lactobacillus rhamnosus GG probiotics and to intensive therapy with traditional (metronidazole, vancomycin) and experimental (rifaximin, nitazoxanide) antibiotics despite its apparent antimicrobial-susceptible phenotype. After excluding other infectious causes of diarrhea and inflammatory bowel disease, we designed a protocol to safely administer fecal bacteriotherapy via a temporary nasogastric tube. We demonstrated for the first time that fecal transplantation is practical and effective for treating relapsing CDI in a young child. We recommend that this strategy be reserved for complicated cases of CDI that fail conventional therapy until randomized studies can confirm the safety and effectiveness of fecal bacteriotherapy in children.

Effectiveness of a guideline to reduce vancomycin use in the neonatal intensive care unit.

Background: The Centers for Disease Control and Prevention recommend hospitals develop guidelines for the appropriate use of vancomycin as part of comprehensive antimicrobial stewardship. The objective of this study was to evaluate the effectiveness and safety of a guideline to restrict vancomycin use in the neonatal intensive care unit (NICU). Methods: A vancomycin use guideline was introduced in 2 tertiary care NICUs with low incidences of methicillin-resistant Staphylococcus aureus infections. We compared all infants >72 hours of age who were evaluated for late-onset infection before and after implementation of this guideline. Results: Vancomycin start rates were reduced from 6.9 to 4.5 per 1000 patient-days (35% reduction; P = 0.01) at Brigham and Womens Hospital, and from 17 to 6.4 per 1000 patient-days (62% reduction; P < 0.0001) at Massachusetts General Hospital. The number of infants exposed to vancomycin decreased from 5.2 to 3.1 per 1000 patient-days (40% reduction; P = 0.008) at Brigham and Womens Hospital, and 10.8 to 5.5 per 1000 patient-days (49% reduction; P = 0.009) at Massachusetts General Hospital. Causes of infection, duration of bacteremia, and incidence of complications or deaths attributable to late-onset infection did not change significantly at either institution. Conclusions: Implementation of a NICU vancomycin use guideline significantly reduced exposure of newborns to vancomycin without adversely affecting short-term patient safety. Further studies are required to evaluate the long-term effect of vancomycin restriction on NICU patient safety and microbial ecology, particularly among institutions with higher rates of methicillin-resistant Staphylococcus aureus infections.

Antibodies to PfSEA-1 block parasite egress from RBCs and protect against malaria infection

Novel vaccines are urgently needed to reduce the burden of severe malaria. Using a differential whole-proteome screening method, we identified Plasmodium falciparum schizont egress antigen-1 (PfSEA-1), a 244-kilodalton parasite antigen expressed in schizont-infected red blood cells (RBCs). Antibodies to PfSEA-1 decreased parasite replication by arresting schizont rupture, and conditional disruption of PfSEA-1 resulted in a profound parasite replication defect. Vaccination of mice with recombinant Plasmodium berghei PbSEA-1 significantly reduced parasitemia and delayed mortality after lethal challenge with the Plasmodium berghei strain ANKA. Tanzanian children with antibodies to recombinant PfSEA-1A (rPfSEA-1A) did not experience severe malaria, and Kenyan adolescents and adults with antibodies to rPfSEA-1A had significantly lower parasite densities than individuals without these antibodies. By blocking schizont egress, PfSEA-1 may synergize with other vaccines targeting hepatocyte and RBC invasion. Antibodies in Tanzanian children identify a malaria vaccine candidate that prevents within-host dispersal of blood-stage parasites Progress toward an effective malaria vaccine The history of efforts to develop a malaria vaccine has been long and difficult. Raj et al. probed for molecules produced by this blood parasite that are recognized by natural immune responses of people living in malaria-endemic areas of Africa. One, PfSEA-1, blocked parasite exit from red blood cells. Vaccination experiments with mouse malaria showed almost fourfold reduction in parasitemia; moreover, passive transfer of PfSEA-1 antibodies transferred protection from mouse to mouse. Encouragingly, the presence of PfSEA-1 antibodies correlates with significant protection from severe malaria in children and adolescents from Kenya and Tanzania. Science, this issue p. 871

Diagnostic Utility and Clinical Significance of Naso- and Oropharyngeal Samples Used in a PCR Assay To Diagnose Mycoplasma pneumoniae Infection in Children with Community-Acquired Pneumonia

ABSTRACT PCR assays of naso- and oropharyngeal samples among hospitalized children appear equally effective for the diagnosis of serologically confirmed community-acquired mycoplasmal pneumonia. However, the combination of results from both sites yields optimal sensitivity (57%), specificity (98%), and positive (92%) and negative (82%) predictive values when compared with Mycoplasma pneumoniae enzyme-linked immunosorbent assay.

A Novel L-ficolin/Mannose-binding Lectin Chimeric Molecule with Enhanced Activity against Ebola Virus

Ebola viruses constitute a newly emerging public threat because they cause rapidly fatal hemorrhagic fevers for which no treatment exists, and they can be manipulated as bioweapons. We targeted conserved N-glycosylated carbohydrate ligands on viral envelope surfaces using novel immune therapies. Mannose-binding lectin (MBL) and L-ficolin (L-FCN) were selected because they function as opsonins and activate complement. Given that MBL has a complex quaternary structure unsuitable for large scale cost-effective production, we sought to develop a less complex chimeric fusion protein with similar ligand recognition and enhanced effector functions. We tested recombinant human MBL and three L-FCN/MBL variants that contained the MBL carbohydrate recognition domain and varying lengths of the L-FCN collagenous domain. Non-reduced chimeric proteins formed predominantly nona- and dodecameric oligomers, whereas recombinant human MBL formed octadecameric and larger oligomers. Surface plasmon resonance revealed that L-FCN/MBL76 had the highest binding affinities for N-acetylglucosamine-bovine serum albumin and mannan. The same chimeric protein displayed superior complement C4 cleavage and binding to calreticulin (cC1qR), a putative receptor for MBL. L-FCN/MBL76 reduced infection by wild type Ebola virus Zaire significantly greater than the other molecules. Tapping mode atomic force microscopy revealed that L-FCN/MBL76 was significantly less tall than the other molecules despite similar polypeptide lengths. We propose that alterations in the quaternary structure of L-FCN/MBL76 resulted in greater flexibility in the collagenous or neck region. Similarly, a more pliable molecule might enhance cooperativity between the carbohydrate recognition domains and their cognate ligands, complement activation, and calreticulin binding dynamics. L-FCN/MBL chimeric proteins should be considered as potential novel therapeutics.

High-Dose Mannose-Binding Lectin Therapy for Ebola Virus Infection

Mannose-binding lectin (MBL) targets diverse microorganisms for phagocytosis and complement-mediated lysis by binding specific surface glycans. Although recombinant human MBL (rhMBL) trials have focused on reconstitution therapy, safety studies have identified no barriers to its use at higher levels. Ebola viruses cause fatal hemorrhagic fevers for which no treatment exists and that are feared as potential biothreat agents. We found that mice whose rhMBL serum concentrations were increased ≥7-fold above average human levels survived otherwise fatal Ebola virus infections and became immune to virus rechallenge. Because Ebola glycoproteins potentially model other glycosylated viruses, rhMBL may offer a novel broad-spectrum antiviral approach.

Pharmacodynamics and Bactericidal Activity of Moxifloxacin in Experimental Escherichia coli Meningitis

ABSTRACT Moxifloxacin, an 8-methoxyquinolone with broad-spectrum activity in vitro, was studied in the rabbit model of Escherichia colimeningitis. The purposes of this study were to evaluate the bactericidal effectiveness and the pharmacodynamic profile of moxifloxacin in cerebrospinal fluid (CSF) and to compare the bactericidal activity with that of ceftriaxone and meropenem therapy. After induction of meningitis, animals were given single doses of 10, 20, and 40 mg/kg or divided-dose regimens of 5, 10, and 20 mg/kg twice, separated by 6 h. After single doses, the penetration of moxifloxacin into purulent CSF, measured as percentage of the area under the concentration-time curve (AUC) in CSF relative to the AUC in plasma, was approximately 50%. After single doses of 10, 20, and 40 mg/kg, the maximum CSF concentration (Cmax) values were 1.8, 4.2, and 4.9 μg/ml, respectively; the AUC values (total drug) were 13.4, 25.4, and 27.1 μg/ml · h, respectively, and the half-life values (t½) were 6.7, 6.6, and 4.7 h, respectively. The bacterial killing in CSF for moxifloxacin, calculated as the Δlog10 CFU per milliliter per hour, at 3, 6, and 12 h after single doses of 10, 20, and 40 mg/kg were −5.70, −6.62, and −7.02; −7.37, −7.37, and −6.87; and −6.62, −6.62, and −6.62, respectively, whereas those of ceftriaxone and meropenem were −4.18, −5.24, and −4.43, and −3.64, −3.59, and −4.12, respectively. The CSF pharmacodynamic indices of AUC/MBC and Cmax/MBC were interrelated (r = 0.81); there was less correlation withT > MBC (r = 0.74). In this model, therapy with moxifloxacin appears to be at least as effective as ceftriaxone and more effective than meropenem therapy in eradicatingE. coli from CSF.

Value of cerebrospinal fluid leukocyte aggregation in distinguishing the causes of meningitis in children.

BACKGROUND Current laboratory tests often cannot distinguish between bacterial and aseptic meningitis rapidly and accurately. The ability to make a prompt diagnosis has important implications for the management and outcome of children with meningitis. The observation that leukocytes aggregate in the cerebrospinal fluid (CSF) has been previously reported, and it has been advocated as a reliable method to distinguish the causes of meningitis in children. OBJECTIVE To investigate the utility of CSF leukocyte aggregation as a screening test to distinguish between bacterial and aseptic meningitis. METHODS We compared the clinical and laboratory indices of 109 prospectively enrolled patients with meningitis (67 bacterial, 23 viral, 19 undefined etiology) and evaluated the validity of the CSF leukocyte aggregation test. The predefined leukocyte aggregation scores (LAS) were compared among the types of meningitis, and correlations with other markers of inflammation were calculated. RESULTS The median LAS was significantly higher (P < 0.001) in the bacterial (32.1%; range, 0 to 84.1%) than in the viral (0%; range, 0 to 16.6%) or undefined (0%; range, 0 to 20.7%) groups. The optimal sensitivity of the leukocyte aggregation test, 98.5 to 92.5%, was demonstrated with LAS values of 0 to 3%. The corresponding specificity was 64.3 to 88.1%. The peripheral white blood cell (WBC) count, serum C-reactive protein, CSF WBC count, blood culture, CSF Gram stain and CSF culture were inferior to the LAS as screening tests when compared individually. The LAS was as effective as CSF protein, TNF-alpha, IL-1-beta, IL-6 and IL-8 to predict bacterial meningitis. In a logistic regression model that included routine laboratory tests, the best predictor of bacterial meningitis was the LAS (odds ratio, 1.6 to 3.7). Significant correlations were demonstrated between the LAS and CSF protein, CSF WBC count, IL-1-beta, IL-6 and IL-8. Duration of symptoms before diagnosis, pretreatment with antibiotics, HIV-1 infection status and CSF red blood cell count did not significantly alter the LAS. CONCLUSIONS There is no single test to diagnose the etiology of meningitis in children promptly and accurately. The finding of leukocyte aggregation in CSF might be of value as a sensitive adjunctive screening tool for the timely diagnosis of bacterial meningitis, recognizing that it has low specificity and potential practical limitations.