Genomic description of acquired fluconazole- and echinocandin-resistance in patients with serial Candida glabrata isolates.

Candida glabrata is one of the most common causes of systemic candidiasis, often resistant to antifungal medications. To describe the genomic context of emerging resistance, we conducted a retrospective analysis of 82 serially collected isolates from 33 patients from population-based candidemia surveillance in the United States. We used whole-genome sequencing to determine the genetic relationships between isolates obtained from the same patient. Phylogenetic analysis demonstrated that isolates from 29 patients were clustered by patient. The median SNPs between isolates from the same patient was 30 (range: 7-96 SNPs), while unrelated strains infected four patients. Twenty-one isolates were resistant to echinocandins, and 24 were resistant to fluconazole. All echinocandin-resistant isolates carried a mutation either in the FKS1 or FKS2 HS1 region. Of the 24 fluconazole-resistant isolates, 17 (71%) had non-synonymous polymorphisms in the PDR1 gene, which were absent in susceptible isolates. In 11 patients, a genetically related resistant isolate was collected after recovering susceptible isolates, indicating in vivo acquisition of resistance. These findings allowed us to estimate the intra-host diversity of C. glabrata and propose an upper boundary of 96 SNPs for defining genetically related isolates, which can be used to assess donor-to-host transmission, nosocomial transmission, or acquired resistance. IMPORTANCE In our study, mutations associated to azole resistance and echinocandin resistance were detected in Candida glabrata isolates using a whole-genome sequence. C. glabrata is the second most common cause of candidemia in the United States, which rapidly acquires resistance to antifungals, in vitro and in vivo.

Revascularization of the upper extremity in a preterm infant: a case report and review of the literature.

Upper extremity arterial injuries in preterm infants are usually of iatrogenic origin. Current microsurgical techniques permit extremity revascularization in these patients. The authors report the microsurgical repair of a 0.7-mm brachial artery in a 940-g preterm infant. The preterm infant warrants special consideration due to physiological immaturity. Rapid fluid shifts, a relative polycythemia, and the potential for low cardiac output states increase the risk for vascular thrombosis. Systemic heparinization is contraindicated in this population due to the risk of intraventricular hemorrhage. Optimization of various physiological variables should reduce the risk of thrombosis.

Microvascular transfer of the combined gastrocnemius-soleus muscle flap in the rat.

The combined gastrocnemius-soleus (G-S) muscle flap is a new research model for use in microvascular free tissue transfer in rats. Harvest of the combined G-S muscle flap is straightforward technically, and its increased size makes it particularly useful for biochemical and morphological study. A single G-S muscle complex accounts for approximately 1% of a rat's total body weight (261.67 +/- 21.60 g). It can be used as a free muscle flap based on its large femoral pedicle (artery diameter, 0.9 +/- 0.1 mm; vein diameter, 1.6 +/- 0.1 mm). Eight G-S muscle flaps were harvested as free muscle flaps and transferred to a recipient femoral site. The flaps were explored 24 hours after revascularization and assessed for viability by visual inspection of the muscle and vascular pedicle, as well as by fluorescein injection. A survival rate of 100% (8 of 8) was noted at 24 hours. This flap is reliable, easily harvested, and provides additional muscle bulk for microvascular research.

Reduction of reperfusion injury edema with corticotropin-releasing factor (CRF): a pilot study.

Regulation of capillary permeability with ischemic reperfusion injury is a complex interaction between vascular endothelium and circulating blood factors. Corticotropin-releasing factor (CRF), a 41 -amino acid peptide CNS neurotransmitter known to modulate the pituitary-adrenal axis during stress response, has been shown to affect capillary leakage after thermal injury in peripheral tissue. When administered prior to ischemic reperfusion injury in a pedicled rat hindlimb model, CRF (80 mcg/kg) reduced limb weight gain to approximately 50% of saline control, suggesting a role for CRF in control of vascular permeability. The characteristics of CRF are reviewed.

Successful twin pregnancy and delivery following free rectus abdominis muscle flap at 15 weeks gestational age.

Although a vertical rectus abdominis flap would not have been selected for reconstruction of the patient's defect had her pregnancy been detected preoperatively, the present case does demonstrate the remarkable resiliency and integrity of the anterior abdominal wall after rectus abdominis muscle flap surgery. Meticulous closure of the abdominal wall is of utmost importance in maintaining abdominal wall competence. Although the merits of muscle splitting techniques and the use of mesh are beyond the scope of this report, there is no evidence that modification of technique should be performed in the patient considering future pregnancy. Our case supports other reports that rectus abdominis flap surgery is not a contraindication to future pregnancy. Intuitively waiting at least 1 year, as recommended by Chen et al., seems reasonable, although the present case demonstrated a successful pregnancy and delivery of twins after a vertical rectus abdominis flap was harvested during pregnancy.

Carbon dioxide laser ablation of anogenital condyloma acuminata in pediatric patients.

The treatment of anogenital condyloma acuminata in pediatric patients is difficult, with a wide range of treatment strategies that yield variable success. Treatment regimens must consider the patient age, and etiology, location, and severity of lesions. We report our experience using the carbon dioxide (CO2) laser to ablate these lesions. A retrospective review of a single surgeon's series of 17 consecutive patients was performed. A staging system was developed and used to document the extent of disease as related to prognosis, recurrence rates, and treatment options. There were 17 patients (5 males and 12 females). Eleven patients (65%) were treated after failing previous treatment. Perianal disease was noted in 14 of 17 patients (82%). No patients presented with stage I disease, 7 patients (41%) presented with stage II, 5 patients (29%) presented with stage III, and 5 patients (29%) presented with stage IV disease. Recurrence occurred in 4 of 17 patients (23%). Persistent disease occurred in 1 patient (6%). CO2 laser vaporization of anogenital condyloma acuminata in pediatric patients is safe, relatively atraumatic to the child, and associated with favorable recurrence rates. A staging scheme has been proposed in predicting recurrences, complications, and guiding therapy.

Variations in the vascular pedicle of the rat gracilis muscle flap.

The rat gracilis muscle is a reliable model for microsurgical research. Exploration of 364 inguinal regions and preparation of 80 free gracilis muscle flaps in Sprague-Dawley and Lewis rats revealed six distinct origins of the dominant arterial pedicle and four variations of the venous pedicle. Popliteal, external iliac, and truncus pudendoepigastricus associations are described for the first time in this study. The total incidence of atypical variation of arterial pedicle origin is 20.7 percent. The most common origin of muscular branch arteries are the superficial epigastric artery (53.3 percent), femoral artery (22.0 percent), popliteal artery (14.6 percent), and the external iliac arteries (5.5 percent). Muscular branch veins drain most frequently into the femoral vein (79.1 percent). Gracilis muscle flaps prepared from rats weighing 329 +/- 45 g weighed an average of 0.64 +/- 0.13 g, and their volume averaged 0.87 +/- 0.12 ml. Muscular branch artery and vein diameters (external) averaged 0.21 +/- 0.05 mm and 0.54 +/- 0.06 mm, respectively. This new anatomic information is essential for reproducible microsurgery research using the rat gracilis muscle.

Subungual keratoacanthoma masquerading as a chronic paronychia.

Subungual keratoacanthoma is a rare, benign tumor of the digits. Patients present with progressive fusiform swelling, erythema, and tenderness, usually affecting a single digit on the radial side of the hand. A cup-shaped lytic lesion of the distal phalanx is a uniform finding on radiography. Delay in diagnosis and misdiagnosis are common because of the rarity of the lesion and difficulties with histological differentiation from subungual squamous cell carcinoma. Accurate diagnosis requires a high index of suspicion, a careful history, and histological evaluation. The natural progression of the disease appears to be continued growth with ongoing destruction of the distal phalanx. Proper treatment involves surgical removal of the mass by curettage and close follow-up for at least 2 years to monitor for local recurrence.

Improved sternal fixation in the correction of pediatric pectus excavatum.

Pectus excavatum is the most common congenital chest wall deformity; it is manifest by abnormal costal cartilage segments causing sternal depression and rotation. The most popular repair involves resection of the abnormal costal cartilage segments, followed by sternal osteotomy and fixation to the adjacent chest wall. Methods of sternal fixation have included metal bars and struts, K-wires, pins, sutures, synthetic mesh, vascularized rib struts, and large AO plates, but they have not included more recent rigid fixation technology. Current rigid fixation techniques allow incorporation of malleable, low-profile reconstruction plates that can be contoured to correct the three-dimensional aspect of these defects. Three pediatric patients are presented in whom plate fixation was utilized for sternal fixation. Current reconstruction plate technology provides an improved fixation alternative for the correction of congenital chest wall anomalies, as well as offering similar potential advantages for traumatic, oncologic, and reconstructive surgery of the thorax.

Quantitation of platelet deposition on human arteries: assessment of the disparity between results obtained with 111indium (111In)-labelling versus scanning electron microscopy.

Quantitation techniques for measuring platelet deposition (PD) to vessel surfaces are important to an understanding of thrombogenesis. In previous studies, scanning electron microscopy (SEM) has been shown to indicate a lower extent of PD than platelet 111In-scintigraphy. Part of this disparity may be explained by nonspecific binding of 111In to the vessel surface during perfusion, or loss of adherent 111In-labelled platelets by lysis or dissociation from the surface during specimen preparation for SEM. To assess whether these independent processes occur, we used a previously described human placental artery (HPA) perfusion model to quantify vessel 111In retention. Of the total 111In that bound to the vessel surface during perfusion, 77 +/- 42% (N = 9) was platelet associated 111In (111In-labelled platelets) and 23 +/- 19% (N = 9) was non-platelet associated 111In (nonspecific binding). After specimen fixation, 67 +/- 32% (N = 9) of the initial total surface 111In remained. This decrease is due to dissociation of both adherent 111In-labelled platelets, and nonplatelet associated 111In. After fixation, 57 +/- 34% (N = 9) of the initial total surface 111In remained as 111In-labelled platelets and 10 +/- 13% (N = 9) remained as nonplatelet associated 111In. Fixation caused no measurable lysis of platelets. These data suggest that PD may be overestimated by 111In-scintigraphy because of nonspecific binding of 111In and underestimated by SEM because of dissociation of adherent platelets during specimen preparation for SEM.

Laser-assisted microvascular anastomosis of human adult and placental arteries with expanded polytetrafluoroethylene microconduit.

Laser-assisted microvascular anastomoses can be performed more quickly than sutured anastomoses, yet manifest similar patency rates and tensile strength. This study was undertaken to determine if in vitro laser-assisted microvascular anastomoses could be created between human adult arteries (anterior tibial arteries), human placental arteries, and expanded polytetrafluoroethylene microconduits. A CO2 laser was applied in single or continuous bursts with a matrix of variables encompassing power P = 80 to 160 mW, spot size SS = 150 to 500 microns, and exposure time EXP = 1.0-second continuous exposure (n = 2 each composite setting). The endpoints measured to assess the ability to laser-weld vessels were morphologic appearance by scanning electron microscopy and bursting strength. Scanning electron microscopy revealed apparent fusion of human placental arteries and human adult arteries to expanded polytetrafluoroethylene microconduits at settings of P = 130 mW, SS = 300 microns, and EXP = 1.0 second, though bursting pressure at all settings was less than 10 mmHg. Laser-assisted microvascular anastomoses of human placental artery to human placental artery and human adult artery to human adult artery were successful at this setting, though bursting pressures of anastomoses incorporating placental vessels were significantly weaker than those created with adult tissue. The relative weakness of laser-assisted microvascular anastomoses incorporating placental arteries might be explained by qualitative or quantitative differences in vessel wall collagen, as seen in fetal tissue, and deserves further characterization.

The effect of microvascular anastomosis configuration on initial platelet deposition.

The propensity for platelets to bind at a native vessel anastomosis is thought to be related to subendothelial exposure, the presence of suture material, and local flow disturbances. By using an artificial microvascular graft to artificial microvascular graft anastomosis model that mimics the geometry and topography of a native microvascular anastomosis but which eliminates the endothelial and subendothelial contributions, the influence of the normal anastomotic configuration alone on initial platelet deposition was measured. Anastomotic and immediate downstream platelet deposition was not augmented by the presence of the anastomotic configuration alone. This suggests that the enhanced initial platelet deposition in the region of a native vessel microanastomosis is primarily related to the presence of injured endothelium and exposed subendothelium rather than to flow disturbances.

Initial platelet deposition at the human microvascular anastomosis: effect on downstream platelet deposition to intact and injured vessels.

Initial platelet deposition (PD) in and around the region of a small-vessel anastomosis may set the stage for thrombosis and tissue loss. To study this problem, a human vessel model (human placental artery, HPA) has been designed to mimic the vascular injuries attendant on clinical microsurgery. To perform these studies, dissected lengths of human placental artery were treated to provide the following four types of injury: group I: control, dissected but otherwise uninjured (N = 5); group II: distal portion of vessel endothelium removed (N = 5); group III: central anastomosis, distal endothelium intact (N = 7); and group IV: central anastomosis, distal endothelium removed (N = 4). Vessels were perfused with 25 ml human whole blood for 17 +/- 5 s at an average shear rate of 536 s-1. Vessels in groups I to IV were segmented at 2-cm intervals, and the number of 111In-labeled platelets was measured. Data from the following groups of exposure zones were pooled and analyzed: endothelium intact, endothelium absent, anastomosis present, postanastomosis with endothelium intact, and postanastomosis with endothelium absent. Significant numbers of platelets were found to attach to intact endothelium, indicating that ischemia and microsurgical handling may augment platelet deposition to otherwise uninjured vessels. A similar degree of platelet deposition was measured after exposure of the subendothelium and perfusion, indicating that superficial subendothelial exposure in the absence of an additional prothrombotic stimulus may lead to no greater platelet deposition than occurs on slightly injured endothelium alone. Platelet deposition at anastomoses was strikingly elevated, although the anastomosis had no additive effect on platelet deposition to downstream endothelium.(ABSTRACT TRUNCATED AT 250 WORDS)

Improved chest wall fixation for correction of pectus excavatum.

Pectus excavatum, the most common congenital chest wall abnormality, is manifested by deformity of the costal cartilages resulting in a depressed and often rotated sternum. Although there are conflicting data to support and reject the concept that physiologic improvement can be a consequence of surgical repair, correction is frequently indicated for aesthetic improvement alone. The most popular current repair involves resection of abnormal costal cartilages, sternal osteotomy and mobilisation, followed by fixation of the sternum in the corrected position. Improved fixation techniques have evolved, but generally have not employed current concepts of rigid fixation. The correction of pectus excavatum using reconstruction plates incorporates the benefits of rigid fixation, while allowing custom chest wall contouring and sternal reorientation. Reconstruction plate fixation of the sternum should be considered during correction of pectus excavatum in adult and adolescent patients.

Vasospasm and platelet deposition in human arteries: effects of topical methylene blue.

Vasodilation of small blood vessels is controlled in part by the endothelium-derived relaxing factor (EDRF), which also inhibits platelet adhesion. Methylene blue (MB), which is occasionally applied directly to blood vessels during microsurgery to provide orientation and prevent torsion, is an irreversible inhibitor of the effects of endothelium-derived relaxing factor and may thereby augment both vasospasm and platelet responses. We have investigated the effects of the extravascular adventitial application of methylene blue on platelet deposition to human placental arteries (HPA) in the presence and absence of surgically induced vasospasm. A trend toward increased platelet deposition to human placental arteries was seen in each group but did not reach significance. The degree of platelet deposition to control human placental arteries suggests that the effects of methylene blue on platelet deposition may be dwarfed by the effects of surgical trauma and ischemia.

Platelet deposition on polyethylene. Dependence of second layer platelet attachment on von Willebrand's disease plasma.

The role of von Willebrand's factor (VWF) was examined in platelet deposition on polyethylene by exposing 3/16 inch diameter discs of polyethylene to 111In labeled platelets re-suspended in citrated blood from normal and severe von Willebrand's disease donors. Discs were oscillated for 30 and 60 min, washed, fixed, counted for 111In, and examined by scanning electron microscopy (SEM). Total platelet deposition in the 0% VWF group was significantly greater than control discs at 30 min (7.0 x 10(6) +/- 0.5 compared with 3.29 x 10(6) +/- 0.6 platelets/cm2). The SEM examination revealed patches of spread platelets, with platelets adhering to platelets in controls. A dense, uniform monolayer of platelets was found on the 0% VWF discs. At 60 min, control discs had significant increases in the platelet deposition with SEM examination revealing uniform coverage of the disc with additional platelets.

Inhibition of platelet retention on artificial microvascular grafts with monoclonal antibodies and a high-affinity peptide directed against platelet membrane glycoproteins.

Rapid occlusion of artificial microvascular grafts (AMGs; less than or equal to 2-mm diameter) by platelet-rich thrombi prevents the clinical use of AMGs in cardiac, vascular, and plastic surgery. Since present antiplatelet agents are unable to assure AMG patency, we have studied the role of specific platelet membrane-glycoprotein blockade on platelet retention by AMGs. In a customized in vitro perfusion chamber, retention on polytetrafluoroethylene (PTFE) AMGs (1.0-mm i.d.) of indium-111-labeled platelets in human whole blood was measured in the presence and absence of inhibitors. Specific blockade of platelet membrane glycoprotein (Gp) IIb/IIIa was achieved using monoclonal antibody 10E5 (10 micrograms/ml) and the peptide GRGDS (Gly-Arg-Gly-Asp-Ser, 0.75 mM). These inhibited 98% and 35%, respectively, of platelet retention under circumstances in which aspirin (7 mM) and dextran (4 mg/ml) inhibited 19% and 18%, respectively, of platelet retention. Nonspecific immunoglobulin G F(ab')2 (10 micrograms/ml) and nonspecific peptide (GGDA; Gly-Gly-Asp-Ala, 0.75 mM), used as control reagents, were ineffective in this setting. Monoclonal antibody 6D1 (10 micrograms/ml), which blocks platelet membrane GpIb, prevented 82% of platelet retention on PTFE. These doses of 10E5 and GRGDS completely inhibited platelet aggregation in response to 20 microM ADP, and the dose of 6D1 completely inhibited ristocetin-induced platelet agglutination. The aspirin dose prevented the second phase of ADP-induced aggregation. These data indicate that not only does initial platelet adhesion to PTFE require GpIIb/IIIa but also that GpIb plays a significant role in the early stages of platelet retention on PTFE AMGs.

Extremity replantation.

Although the success of replantation and revascularization is in part related to the length of the ischemic interval, it is important to bear in mind that microsurgical intervention must be given appropriate priority relative to the patient's other injuries. Very few patients die as a result of upper extremity trauma. However, the economic, psychologic, and physical debility of a chronic hand injury will be the ultimate handicap in patients who survive multisystem trauma. Therefore, management of the injured extremity has an important place in the overall scheme of trauma care.