First Report of Capsicum chlorosis virus Infecting Waxflower (Hoya calycina Schlecter) in the United States.

In February 2013, an ornamental waxflower (Hoya calycina Schlecter) with leaves displaying concentric chlorotic and necrotic rings surrounding sunken, necrotic lesions typical of tospovirus infection was observed at a community garden in Honolulu, HI. Symptomatic leaf tissue tested negative for Tomato spotted wilt virus (TSWV), a common tospovirus in Hawaii, using a TSWV ImmunoStrips (AgDia, Elkhart, IN) assay following the manufacturer's instructions. Double-stranded RNAs were isolated from a symptomatic leaf and reverse transcribed using random primers (2). The cDNA was then used as template in a universal tospovirus PCR assay using primers gL3637 and gL4435c, which amplify sequences of the L segment encoding the RNA-dependent RNA polymerase of tospoviruses (1). An ~800-bp product was amplified and cloned using pGEM-T Easy (Promega, Madison, WI). Three clones were selected and found to be identical by dye-terminator sequencing performed at the University of Hawaii's Advanced Studies in Genomics, Proteomics, and Bioinformatics laboratory. Following primer sequence trimming, the 773-bp sequence (GenBank Accession No. KF030938) was found to be 97, 88, and 87% identical to Capsicum chlorosis virus (CaCV; a tentative species in the family Bunyaviridae, genus Tospovirus) strains Ch-Har (GU199334), TwTom1 (HM021140), and AIT (DQ256124), respectively. To confirm the presence of CaCV, the cDNA was also used as template in a universal tospovirus PCR assay with primers 3'T12 and TsMCR2 which amplify a region of the S segment of tospoviruses (3). The amplification product from this assay was cloned and sequenced as described above and found to be 93 to 98% identical to CaCV nucleotide sequences present in GenBank. Attempts to detect the CaCV strain in waxflower using a watermelon silver mottle virus and groundnut bud necrosis virus triple antibody sandwich ELISA (AgDia) were unsuccessful. No other plants in the community garden had typical tospovirus-like symptoms; however, samples from tomato (Solanum lycopersicum L.; two samples), chili pepper (Capsicum spp.; four samples), eggplant (Solanum melongena L.; one sample), and passionfruit (Passiflora edulis Sims; one sample) with virus-like symptoms were collected from the garden and had RNA isolated using a NucleoSpin RNA II kit (Macherey-Nagel, Bethlehem, PA). No tospoviruses were detected in any of these samples with the RT-PCR assay using primers gL3637 and gL4435. The waxflower plant infected with CaCV was immediately removed by community garden members and destroyed, preventing any additional serological or biological assays to be performed. CaCV is transmitted by several species of thrips, including Thrips palmi, which is present in Hawaii. Waxflower is not native to Hawaii and it is unclear whether CaCV entered Hawaii in this plant or whether it was infected by viruliferous thrips. A survey for CaCV in known hosts is essential to determine the geographic distribution of CaCV in Hawaii, as this virus poses a considerable threat to tomato, chili pepper, and phalaenopsis orchid production in Hawaii and the United States. References: (1) F.-H. Chu et al. Phytopathology 91:361, 2001. (2) M. J. Melzer et al. Virus Genes 40:111, 2010. (3) M. Okuda and K. Hanada. J. Virol. Methods 96:149, 2001.

Current concepts of bone healing.

The intent of this article is to present the current understanding of fracture repair and offer investigative evidence to the use of exogenous growth factors enhancing fracture healing. The authors believe that exogenous growth factors have tremendous clinical applications and will continue to influence bone healing in the future. With the expanding knowledge of the intricate molecular and cellular biology of fracture repair being realized, surgeons will be able to enhance and ensure healing of surgical osteotomies, fractured bone ends, or delayed unions. This field of medicine is continuously growing and the possibilities seem endless.

Gunshot wounds to the lower extremity. A comprehensive review.

A thorough knowledge of the mechanisms by which penetrating projectiles disrupt tissue can assist the physician in evaluating and treating the wound in a rational manner. Projectile mass, velocity, shape, construction, and the characteristics and anatomic constraints of the tissue penetrated determine the amount, type, and location of tissue disruption. The possibility of additional injuries caused by secondary missiles and cavitation must be understood to distinguish between conservative versus surgical care of the patient. Adherence to the principles of treatment discussed aids the physician in judgment when treating gunshot wound victims. Furthermore, the authors present a classification system to facilitate the identification and treatment of lower-extremity gunshot wounds. Regardless of the expertise in understanding wound ballistics, the unknown and unexpected variables create a challenge in treating the gunshot victim. A surgeon who believes in wide excision of tissue from all sides of the wound path in any high-velocity wound probably does more harm than that done by the bullet alone. A surgeon treating gunshot wound victims should follow the dictum: "Treat the wound, not the weapon."

Pilon fractures of the distal tibia.

Pilon fractures are one of the most complicated injuries confronting the lower extremity traumatologist. When faced with the pilon fracture, the surgeon must adhere to the principles of anatomic reduction and rigid stabilization to achieve early range of motion. Open reduction and internal or external fixation or a combination of fixation techniques, expertly placed, ensure the best possible outcome with the least complications. A functional lower extremity following a pilon fracture is possible with meticulous evaluation and repair.

Whole blood tissue factor procoagulant activity is elevated in patients with sickle cell disease.

We developed a simple assay for the measurement of tissue factor procoagulant activity (TF PCA) in whole blood samples that avoids the need for mononuclear cell isolation. This method combines convenience of sample collection and processing with a high degree of sensitivity and specificity for TF. Using this method, we have determined that TF PCA is detectable in whole blood samples from normal individuals, which is itself a novel observation. Essentially all PCA could be shown to be localized in the mononuclear cell fraction of blood. Compared with controls, whole blood TF levels were significantly (P < .000001) elevated in patients with sickle cell disease (SCD), regardless of the subtype of hemoglobinopathy (SS or SC disease). No significant difference in TF PCA was observed between patients in pain crisis compared with those in steady-state disease. Because TF functions as cofactor in the proteolytic conversion of FVII to FVIIa in vitro, it was expected that an increase in circulating TF PCA would lead to an increased in vivo generation of FVIIa. On the contrary, FVIIa levels were actually decreased in the plasma of patients with SCD. Plasma TF pathway inhibitor (TFPI) antigen levels were normal in SCD patients, suggesting that accelerated clearance of FVIIa by the TFPI pathway was not responsible for the reduced FVIIa levels. We propose that elevated levels of circulating TF PCA may play an important role in triggering the activation of coagulation known to occur in patients with SCD. Because TF is the principal cellular ligand for FVIIa, it is possible that increased binding to TF accounts for the diminished plasma FVIIa levels.

The use of implantable venous access devices (IVADs) in children with hemophilia.

PURPOSE: Implantable venous access devices (IVADs), either centrally or peripherally implanted, have become increasingly popular in children with hemophilia to assist in the early treatment of bleeding episodes and in the prevention of arthropathy. Their use has been associated with complications including thrombosis, thrombophlebitis, and infection. We attempted to better define whether the benefits associated with IVADs in this population outweight the associated risks. PATIENTS AND METHODS: We studied the medical records of 35 children from the University of Minnesota's Comprehensive Hemophilia Center who received IVADs between 1992 and 1996. RESULTS: There was no bleeding or thrombophlebitis associated with IVADs in our population. One patient required removal of a central IVAD due to thrombosis. The central IVADs were associated with local infection and bacteremia rates of 3% and 33%, respectively. The rates of local infection and bacteremia associated with peripheral IVADs were both 25%. The majority of infections were cleared with antibiotics, and ports remained intact. Both types of IVADs were associated with a high patient/parent satisfaction. CONCLUSION: Despite being associated with a significant incidence of infection, we believe the benefits of IVADs for children with hemophilia and their families outweigh the risks. Possible explanations for the observed infection rates are discussed.

Treatment of childhood lymphangiomas with interferon-alpha.

PURPOSE: Nonsurgical treatment of lymphangiomas has shown limited efficacy and often carries unacceptable toxicities, demonstrating the need for a more effective, less toxic therapy. PATIENTS AND METHODS: We describe two patients with lymphangiomatosis treated for 12 to 40 months with recombinant interferon-alpha. RESULTS: Both patients demonstrated stabilization or marked improvement of disease, based on clinical and radiologic findings, with minimal toxicity. CONCLUSIONS: The favorable responses to interferon-alpha therapy in these two cases suggest that this is an effective and well-tolerated treatment for lymphangiomas in children.

Ureaplasma urealyticum infections in the perinatal period.

Good evidence indicates the widespread carriage of U. urealyticum among sexually active females. Colonization of the upper genital tract appears to be a risk factor for adverse pregnancy outcomes, though this is not the case for lower-genital-tract colonization. Based on studies in which amniotic fluid obtained from amniocenteses was already positive for U. urealyticum, and its high prevalence in very-LBW neonates, it is likely that the infection is acquired during early pregnancy. A number of observational studies have linked this organism to pulmonary infections, meningeal infections, and bacteremias, particularly in LBW neonates. It is difficult, however, to separate the morbidity that is directly attributable to infection with the organism from morbidity owing to extreme prematurity. Problems in measuring the burden of illness resulting from this organism are compounded by difficulties in its diagnosis. The organism's fastidious nature prevents many laboratories from isolating it from specimens. Rapid and practical methods for identifying the organism are urgently needed. These need to be followed by RCTs to determine if outcomes of pregnant women and babies with various conditions, from whom the organism has been isolated, can be improved through treatment with antimicrobial agents.

Relationship between Symptom Development and Actual Sites of Infection in Leaves of Anthurium Inoculated with a Bioluminescent Strain of Xanthomonas campestris pv. dieffenbachiae.

The infection process of bacterial blight of anthurium was monitored with a bioluminescent strain of Xanthomonas campestris pv. dieffenbachiae. The relationship between symptom expression on infected leaves (assessed visually) and the extent of bacterial movement within tissues (evaluated by bioluminescence emission) varied among anthurium cultivars. In several cultivars previously considered susceptible on the basis of symptom development alone, bacterial invasion of leaves extended far beyond the visually affected areas. In other cultivars previously considered resistant, bacterial invasion was restricted to areas with visible symptoms. In three cultivars previously considered resistant, leaves were extensively invaded by the bacterium, and yet few or no symptoms were seen on infected leaves. The pathogen was consistently recovered from leaf sections emitting bioluminescence but not from sections emitting no light. At an early stage of infection, no significant differences in the percentages of infected areas as determined by visual assessment were observed in any of the cultivars. However, differences among cultivars were detected by bioluminescence as the disease progressed, because bacterial invasion was not always accompanied by symptom expression. In susceptible cultivars, the advancing border of infection was 5 to 10 cm inward from the margins of the visible symptoms and often reached to the leaf petiole even when symptoms were visible in <10% of the total leaf area. Comparisons of anthurium cultivars in which a nondestructive method was used to quantify the severity of leaf infection by a bioluminescent pathogen have enabled us to evaluate susceptibility and resistance to bacterial blight accurately. Such evaluations will be of importance in breeding resistant cultivars for disease control.

Improved detection of Malassezia species in lipid-supplemented Peds Plus blood culture bottles.

The growth of Malassezia species in BACTEC Peds Plus blood culture bottles was optimized by using various lipid supplements. Palmitic acid (3%, wt/vol) was superior and overcame the inhibitory effect of blood in mock clinical specimens. Palmitic acid (3%) supplementation of Peds Plus bottles may improve recovery of Malassezia species in the BACTEC NR 660.

Characterization of thrombomodulin expression in response to retinoic acid and identification of a retinoic acid response element in the human thrombomodulin gene.

Thrombomodulin (TM) is an essential cofactor for the physiologic activation of the anticoagulant protein C by thrombin. We have observed that the expression of TM mRNA in response to retinoic acid was markedly increased in human U937 monoblast-like cells, and human MEG01 megakaryocyte-like cells, but not in human umbilical vein cells, murine hemangioma cells, human K562 erythroblast-like cells, and murine HSD fibroblast-like cells. TM activity in U937 cells and MEG01 cells was not detectable in untreated cells, but developed rapidly after treatment with retinoic acid. In endothelial cells there was minimal change in TM activity in response to retinoic acid treatment. We have isolated clones for the genes for murine and human TM and have identified potential retinoic acid response elements in the 5'-flanking region of the human gene. In U937 cells the increase in mRNA levels was associated with increased transcription, and transient transfection studies with reporter plasmids demonstrate functional retinoic acid response elements present in the 5'-flanking region of the gene. Deletion of, and mutations introduced into, the potential retinoic acid response element confirm the functional response in transient transfections.

Management of leukemic hyperleukocytosis with hydration, urinary alkalinization, and allopurinol. Are cranial irradiation and invasive cytoreduction necessary?

PURPOSE: Hyperleukocytosis secondary to acute leukemia is a medical emergency. Intracranial hemorrhage often leads to death in this setting. Early efforts to prevent this serious complication have included emergent cranial irradiation, with its associated morbidity when used in the young child. Currently, exchange transfusion and/or leukapheresis are employed to acutely lower the peripheral leukocyte count. PATIENTS AND METHODS: We report three infants with acute leukemia and hyperleukocytosis in whom intravenous hydration, alkalinization, and allopurinol therapy alone produced rapid and dramatic decreases in the peripheral leukocyte count. RESULTS: The maximal decrease in leukocyte count averaged 88% within 70 h of starting conservative management. A fall in leukocyte count to < 100 x 10(9)/L was noted at an average of 15 h following hospitalization. No patient developed complications. CONCLUSIONS: When comparing this approach to exchange transfusion and leukapheresis we find it to be both safe and effective. Children with hyperleukocytosis in association with acute lymphocytic leukemia who present without life-threatening complications of an extremely high leukocyte count can be safely and effectively managed with intravenous hydration, alkalinization, and allopurinol therapy.

False-positive MRI detection of recurrent or metastatic pediatric infratentorial tumors.

Gadolinium-DTPA enhanced MRI is the modality of choice when imaging central nervous system infratentorial tumors in the pediatric population. The detection of a new enhancing lesion following initiation of therapy is typically considered pathognomonic for recurrent or metastatic tumor. We report two pediatric patients with infratentorial central nervous system tumors who demonstrated new areas of enhancement with gadolinium-DTPA enhanced MRI following initiation of multimodality therapy. Both patients had surgical biopsy of the new lesions with histologic review failing to demonstrate viable tumor. These studies suggest caution in considering new enhancing lesions detected by MRI in a child with a brain tumor pathognomonic for new sites of active tumor.

Successful therapy for trilateral retinoblastoma.

Trilateral retinoblastoma, the intracranial malignancy associated with bilateral retinoblastoma, is an uncommon and clinically aggressive malignancy with a uniformly fatal outcome. Three children with newly diagnosed trilateral retinoblastoma were treated with systemic (cyclophosphamide and vincristine) and intrathecal (methotrexate, hydrocortisone, and cytarabine) chemotherapy, as well as craniospinal irradiation (one patient) in addition to therapy of the eye lesions. All three patients have had partial or complete response of the pineal tumors to chemotherapy, with no active disease eight or more years, 33 or more months, and 12 or more months, respectively, after diagnosis of the lesions.

Pneumococcal soft-tissue infections: possible association with connective tissue diseases.

Streptococcus pneumoniae is not a well-recognized cause of soft-tissue infections. In less than 4 years, 12 cases of pneumococcal soft-tissue infection were identified through discussions with infections disease subspecialists in the Philadelphia area. Principal sites of involvement included skin and fascia, tongue, epiglottis, thyroid, brain, and breast. Pneumococcal bacteremia was documented in six cases (50%); in three of these, pneumococci were also cultured from the involved soft tissues. In the cases in which bacteremia was not demonstrated, pneumococci were isolated from the infected sites. Six patients had connective tissue diseases, of which five were diagnosed as systemic lupus erythematosus. Four of these patients were receiving corticosteroids when their infections developed. Two additional patients were HIV-seropositive intravenous drug users. S. pneumoniae may be a more important cause of soft-tissue infections than previously appreciated, especially in patients with connective tissue diseases.

Osteomyelitis and synovitis produced by Mycobacterium marinum in a fisherman.

We report a case of osteomyelitis and synovitis produced by Mycobacterium marinum in the left index finger of a fisherman. A combination of surgical intervention and antimicrobial therapy with minocycline, rifampin, and ethambutol was efficacious.