A method for the improved detection of aerosolized influenza viruses and the male-specific (F+) RNA coliphage MS2.

The detection of aerosolized viruses can serve as an important surveillance and control tool in agriculture, human health, and environmental settings. Here, we adapted an anion exchange resin-based method, initially developed to concentrate negatively charged viruses from water, to liquid impingement-based bioaerosol sampling. In this method, aerosolized viruses are collected in a 20ml liquid sample contained within widely used impingers, BioSamplers (SKC Inc., Eighty Four, PA), and further concentrated via adsorption to an anion exchange resin that is suspended within this liquid. Viral nucleic acids are then extracted from the resin to facilitate molecular analyses through a reduction in the effective sample volume. For this study, various quantities of two negatively charged viruses, type A and type B influenza viruses (FluMist Quadrivalent vaccine) and the male-specific (F+) RNA coliphage MS2 (MS2), were nebulized into a custom-built bioaerosolization chamber, and sampled using BioSamplers with and without anion exchange resin. Compared to direct testing of the BioSampler liquid, detection was improved by 6.77× and 3.33× for type A and type B influenza viruses, respectively, by using the anion exchange resin. For MS2, the anion exchange resin method allowed for an average improvement in detection of 8.26×.

Field-based evaluation of a male-specific (F+) RNA coliphage concentration method.

Fecal contamination of water poses a significant risk to public health due to the potential presence of pathogens, including enteric viruses. Therefore, sensitive, reliable and easy to use methods for the concentration, detection and quantification of microorganisms associated with the safety and quality of water are needed. In this study, we performed a field evaluation of an anion exchange resin-based method to concentrate male-specific (F+) RNA coliphages (FRNA), fecal indicator organisms, from diverse environmental waters that were suspected to be contaminated with feces. In this system, FRNA coliphages are adsorbed to anion exchange resin and direct nucleic acid isolation is performed, yielding a sample amenable to real-time reverse transcriptase (RT)-PCR detection. Matrix-dependent inhibition of this method was evaluated using known quantities of spiked FRNA coliphages belonging to four genogroups (GI, GII, GII and GIV). RT-PCR-based detection was successful in 97%, 72%, 85% and 98% of the samples spiked (106 pfu/l) with GI, GII, GIII and GIV, respectively. Differential FRNA coliphage genogroup detection was linked to inhibitors that altered RT-PCR assay efficiency. No association between inhibition and the physicochemical properties of the water samples was apparent. Additionally, the anion exchange resin method facilitated detection of naturally present FRNA coliphages in 40 of 65 environmental water samples (61.5%), demonstrating the viability of this system to concentrate FRNA coliphages from water.

Concentration of enteric viruses from tap water using an anion exchange resin-based method.

Detecting low concentrations of enteric viruses in water is needed for public health-related monitoring and control purposes. Thus, there is a need for sensitive, rapid and cost effective enteric viral concentration methods compatible with downstream molecular detection. Here, a virus concentration method based on adsorption of the virus to an anion exchange resin and direct isolation of nucleic acids is presented. Ten liter samples of tap water spiked with different concentrations (10-10,000 TCID50/10 L) of human adenovirus 40 (HAdV-40), hepatitis A virus (HAV) or rotavirus (RV) were concentrated and detected by real time PCR or real time RT-PCR. This method improved viral detection compared to direct testing of spiked water samples where the ΔCt was 12.1 for AdV-40 and 4.3 for HAV. Direct detection of RV in water was only possible for one of the three replicates tested (Ct of 37), but RV detection was improved using the resin method (all replicates tested positive with an average Ct of 30, n=3). The limit of detection of the method was 10 TCID50/10 L for HAdV-40 and HAV, and 100 TCID50/10 L of water for RV. These results compare favorably with detection limits reported for more expensive and laborious methods.

Evaluation of an anion exchange resin-based method for concentration of F-RNA coliphages (enteric virus indicators) from water samples.

Enteric viral contaminants in water represent a public health concern, thus methods for detecting these viruses or their indicator microorganisms are needed. Because enteric viruses and their viral indicators are often found at low concentrations in water, their detection requires upfront concentration methods. In this study, a strong basic anion exchange resin was evaluated as an adsorbent material for the concentration of F-RNA coliphages (MS2, Qß, GA, and HB-P22). These coliphages are recognized as enteric virus surrogates and fecal indicator organisms. Following adsorption of the coliphages from 50ml water samples, direct RNA isolation and real time RT-PCR detection were performed. In water samples containing 10(5)pfu/ml of the F-RNA coliphages, the anion exchange resin (IRA-900) adsorbed over 96.7% of the coliphages present, improving real time RT-PCR detection by 5-7 cycles compared to direct testing. F-RNA coliphage RNA recovery using the integrated method ranged from 12.6% to 77.1%. Resin-based concentration of samples with low levels of the F-RNA coliphages allowed for 10(0)pfu/ml (MS2 and Qß) and 10(-1)pfu/ml (GA and HB-P22) to be detected. The resin-based method offers considerable advantages in cost, speed, simplicity and field adaptability.

Evaluation of a simple and cost effective filter paper-based shipping and storage medium for environmental sampling of F-RNA coliphages.

Male specific RNA (F-RNA) coliphages are used as indicators of fecal contamination and for source tracking. However, collecting fecal samples for analysis from remote sites is problematic due to the need for an uninterrupted cold chain to guarantee sample suitability for downstream molecular detection of these coliphages. Here, we investigated the feasibility of using filter paper as a collection and storage vehicle for F-RNA coliphages. Various concentrations (10(1) to 10(4)pfu) of two F-RNA coliphages, MS2 and Qß, were prepared in lambda buffer or a 10% bovine manure slurry, spotted onto filter paper disks, dried, and maintained at 37 °C for up to 37 days. Nucleic acids were extracted from the spotted filter paper disks at 0, 6, 13, and 37 days post inoculation and analyzed by real time RT-PCR. F-RNA coliphages at concentrations of 10(2)pfu/filter paper unit were readily detected, and only a slight decrease in nucleic acid detection was observed over time. Furthermore, the sensitivity of real time RT-PCR detection of the F-RNA coliphage RNA was similar between the developed filter paper sampling methodology and traditional cold storage. These results indicate that filter paper is a suitable storage and transport medium for F-RNA coliphages when refrigeration is not possible.

Escape of the yolk sac: a hypothesis to explain the embryogenesis of gastroschisis.

Gastroschisis is a significant birth defect that in many countries has shown an increased prevalence in recent decades, and the change has affected primarily younger mothers. Despite numerous epidemiological studies no other consistent associated risk factor has been identified. In this paper we review the five main theories related to the pathogenesis of this malformation and outline the reasons why we think none fully explains the embryogenesis of gastroschisis. We briefly present some clinical observations we have made that we consider germane to the pathogenesis and outline a hypothesis that we think can account for the origins of this malformation. Our proposal is that the determining defect in gastroschisis is failure of the yolk sac and related vitelline structures to be incorporated into the umbilical stalk. Otherwise, ventral closure of the lateral abdominal walls occurs normally, thus orphaning the vitelline duct and yolk sac outside both the main body stalk and the abdominal wall. Thus, in addition to the umbilicus, the abdominal wall has a separate perforation through which the midpoint of the gut is attached to the exteriorized vitelline structures. This connection through the ventral wall prevents normal egress of the gut into the umbilical cord during the second month of development and acts as the egress point for the gut resulting in gastroschisis.

Prospective results of a standardized algorithm based on hemodynamic status for managing pediatric solid organ injury.

BACKGROUND: Controversy surrounds the need for ICU admission, prolonged bed rest, and the duration of activity restrictions for children sustaining blunt trauma. Adult literature supports management based on hemodynamic status, not CT grade. STUDY DESIGN: A 3-year prospective study of a standardized management algorithm for hemodynamically normal pediatric patients with blunt liver or spleen injury was performed. Patient selection was based on vital signs, irrespective of injury grade on CT. Patients requiring ICU admission for nonliver or nonspleen injury were excluded. Patients were admitted to a surgical ward with serial hematocrit levels. Discharge occurred 48 hours postinjury if patients had no abdominal tenderness, tolerated a regular diet, and had a stable hematocrit. Patients were allowed noncontact activity, including school, after discharge. Patients were followed up at 1 month with ultrasonographic imaging. RESULTS: Eighty-nine patients sustained blunt liver or spleen injury. Forty-five patients were excluded for other injuries (Glasgow Coma Scale < 13, 32 of 45); the remaining 44 patients had a mean age of 8.9 years (range 2 to 17 years), Injury Severity Score 10.6 (range 4 to 33), liver grade 2.1, and splenic injury grade 2.3. Mechanisms of injury were predominately motor vehicle collisions (59%). All patients were managed nonoperatively without transfusion; 43 of 44 patients completed the algorithm. Mean observation was 55.2 +/- 12.3 hours. One-month followup occurred in 33 of 44 patients, with one complication detected and no delayed bleeding. CONCLUSION: Management of pediatric solid organ injury should be guided by hemodynamic status and not injury grade on CT. Hemodynamically normal children can be safely managed without intensive care monitoring, do not need prolonged hospitalization, and can resume school on discharge.

Presentation of carcinoma in a patient with a previous operation for Hirschsprung's disease.

The usual complications after a definitive pull-through procedure for Hirschsprung's disease include stricture formation, enterocolitis, bowel obstruction, and, occasionally, wound infection. The authors report a case of mucinous adenocarcinoma arising 32 years later at a stricture site from a previous pull-through procedure.

Congenital abdominal aortic aneurysm in the infant: case report and review of the literature.

Congenital abdominal aortic aneurysms are a distinct entity from acquired aortic aneurysms. The authors present the case of a 6-week-old boy with a 6-cm aneurysm involving the abdominal aorta and common iliac arteries. Three other cases of congenital aortic aneurysms are reviewed, and an approach to these rare patients is discussed. J Pediatr Surg 36:657-658.

Necrotizing enterocolitis in infants with very low birth weight.

Necrotizing enterocolitis (NEC) is a disease in which the primary risk factor is prematurity. Despite, and partially as a result of, the tremendous strides neonatal care has taken, it is a major cause of morbidity and mortality of the newborn. The infant with very low birth weight is particularly susceptible, and the management of the condition in this group differs somewhat from other neonates. The outcomes continue to improve, but there are significant sequelae. Prevention, which would be the best "cure," is elusive, in no small part because of the multifactorial nature of the etiology of NEC.

Pediatric laparoscopic biliary tract surgery.

Laparoscopic cholecystectomy is being performed with increasing frequency in children. The authors discuss the presentation, surgical technique, overall results, and potential complications associated with pediatric laparoscopic biliary tract surgery, citing a large personal experience as well as that reported in the literature.

Molecular biology and the pediatric surgeon: definitions and basic methodology.

Molecular biology techniques and their application are becoming increasingly important to the practicing clinician. This article reviews the basics of DNA chemistry and highlights important molecular biology techniques. It will provide a guide for the pediatric surgeon as she/he attempts to integrate this field into everyday practice.

Genetic engineering of immunotoxins.

Immunotoxins, composed of both targeting mechanisms and toxins, hold great promise for the treatment of some cancers and other pathological conditions. Research and manufacture of these drugs use various techniques of molecular biology, some of which are described in this article. A considerable amount of research has focused on ricin, a plant toxin, and its immunoconjugates. Areas currently under evaluation by our laboratory include structure-function studies based on mutational analysis, enhancement of intracellular trafficking, genetic fusion of the targeting and toxic moieties, and the expression of toxin in plant cell culture.

Childhood pleuropulmonary blastoma: caution against nonoperative management of congenital lung cysts.

Pulmonary blastoma is a rare and aggressive malignant tumor that affects children and adults. Recently a 3-year-old boy with a 2-year history of bilateral unilocular pulmonary cysts was transferred for evaluation of a cough and high spiking fever. A chest radiogram showed left pulmonary consolidation with pleural effusion, but thoracentesis was unsuccessful. Computerized tomography (CT) was suggestive of a pulmonary abscess, but CT-guided drainage did not yield any purulent fluid. Percutaneous biopsies were performed, and the cytology showed malignant cells. During thoracotomy, a large tumor involving the left lower lobe and pleural space was found, and a biopsy was performed. A frozen section showed blastemal and mesenchymal components devoid of neoplastic epithelium, consistent with the pleural variant of pulmonary blastoma. A left lower lobectomy, with tumor decortication of the pleural space, achieved total gross tumor removal. The child received aggressive multiagent chemotherapy, and midway through it he underwent elective excision of the opposite lung cyst. It has been 17 months since the lobectomy; he is off chemotherapy and has no evidence of disease. A review of the literature showed that a large number of pediatric pulmonary blastomas are associated with cystic lung disease. Because total tumor removal offers the only chance of a good long-term outcome, surgical excision or close follow-up of pulmonary cysts in children is strongly recommended.

A prospective, randomized trial of short versus long tubes in adhesive small-bowel obstruction.

BACKGROUND: Many cases of acute adhesive small-bowel obstruction (SBO) can be successfully treated with intestinal tube decompression. There is considerable controversy, however, regarding whether a short nasogastric tube (NGT) or a long nasointestinal tube (LT) is the best method of intestinal tube decompression. PATIENTS AND METHODS: A prospective, randomized trial was conducted to compare NGT and LT decompression with respect to the success of nonoperative treatment and morbidity of surgical intervention in 55 patients with acute adhesive SBO. RESULTS: Twenty-eight patients were managed with NGT and 27 with LT. There were 44 cases of partial SBO (23 NGT, 21 LT) and 11 cases of complete SBO (5 NGT, 6 LT). Twenty-one patients ultimately required operation, including 13 managed with NGT (46%) and 8 with LT (30%) (P = 0.16). The mean period between admission and operation was 60 hours in the NGT group versus 65 hours in the LT group. At operation, 3 patients in the NGT group had ischemic bowel that required resection. Postoperative complications occurred in 23% of patients treated with NGT versus 38% of patients treated with LT (P = 0.89). Postoperative ileus averaged 6.1 days for NGT patients versus 4.6 days for LT patients (P = 0.44). There were no deaths. CONCLUSIONS: Patients with adhesive SBO can safely be given a trial of tube decompression upon hospital admission. There was no advantage of one type of tube over the other in patients with adhesive SBO.