Effect of the laccase gene CNLAC1, on virulence of Cryptococcus neoformans.

To assess the relationship between melanin production by Cryptococcus neoformans and virulence on a molecular basis, we asked: (a) is CNLAC1, the laccase structural gene of C. neoformans, expressed in vivo?; (b) can mouse virulence be restored to cnlac1 (Mel-) mutants by complementation with CNLAC1?; and (c) will targeted gene deletion of CNLAC1 decrease virulence for mice? Melanin is produced when cryptococcal laccase catalyzes the oxidation of certain aromatic compounds, including L-dopa, to quinones, which then polymerize to melanin. To assess CNLAC1 transcription, RNA was extracted from C. neoformans in cerebrospinal fluid of infected rabbits. Reverse transcriptase-polymerase chain reaction detected CNLAC1 transcript, indicating that laccase may be produced in the infected host. To assess the effect of CNLAC1 deletion on virulence, a Mel- mutant (10S) was obtained by disruption of the 5' end of the gene. After multiple backcrosses with a parental strain to remove unintended genetic defects introduced by the transformation process, a Mel- progeny was tested and found to be much less virulent for mice than a Mel+ progeny. Another Mel- strain (mel2), obtained from J.C. Edman (University of California at San Francisco, CA), produced CNLAC1 transcript but no detectable melanin. Characterization of this mutant revealed a base substitution in CNLAC1 that changed a histidine to tyrosine in a putative copper-binding site. When this base change was introduced into CNLAC1 by site-directed mutagenesis, it no longer transformed mel2 to Mel+, indicating the importance of this histidine in laccase activity. Complementation of a mel2-derived mutant with CNLAC1 restored the Mel+ phenotype and increased virulence. These results support the concept that the CNLAC1 gene product has a role in virulence.

Long-lasting, specific immunologic unresponsiveness associated with cryptococcal meningitis.

A sensitive radioimmunoassay and an antibody class-specific enzyme-linked immunosorbent assay were used to determine whether patients cured of cryptococcosis responded normally to immunization with cryptococcal capsular polysaccharide (CPS) and type III pneumococcal polysaccharide. 10 normal volunteers and 8 patients who had been cured of cryptococcal meningitis and who had been cured of cryptococcal meningitis and who had no serious underlying diseases were immunized with both antigens. Geometric mean titers to CPS measured by radioimmunoassay were 1:1 in both groups before vaccination, but were 1:3 in patients and 1:119 in controls following immunization (P less than 0.01, Student's t test). Analysis of the class-specific response to immunization with CPS found little anti-CPS IgG or IgA. Geometric mean postvaccination IgM titers were 1:31 in patients and 1:238 in controls (P less than 0.01). Responses to immunization with type III pneumococcal polysaccharide were similar in patients and controls, with IgA, IgM, and IgG mean titers of 1:1129, 1:369, and 1:158 in patients and 1:1504, 1:1039, and 1:163 in controls (P greater than 0.2 for each antibody class). Cured cryptococcal meningitis is often associated with prolonged specific immunologic unresponsiveness.

Galactofuranosyl groups are immunodominant in Aspergillus fumigatus galactomannan.

Aspergillus fumigatus galactomannan was prepared and its structure partially characterized. Galactofuranosyl groups were immunodominant when this polysaccharide antigen was reacted with antibody raised in rabbits.

Differences in Cryptococcus neoformans capsular polysaccharide structure influence assembly of alternative complement pathway C3 convertase on fungal surfaces.

Binding of complement component C3 and Factor B to Cryptococcus neoformans serotypes A through D via the alternative complement pathway was measured in a system containing fresh nonimmune human serum. Serotypes B and C (C. neoformans var. gattii) bound approximately half as many molecules of both complement components as serotypes A and D (C. neoformans var. neoformans). In contrast, removal of xylosyl and glucuronyl side chains from the mannan main chain of capsular polysaccharide by the Smith degradation procedure resulted in binding of similar quantities of C3 to each of the four serotypes. We conclude that the relatively high degree of side chain substitution of capsular polysaccharide from C. neoformans variety gattii contributes to inefficient surface assembly of the alternative pathway C3 convertase. Inefficient binding of alternative pathway complement components to serotypes B and C may contribute to the relative difficulty in successfully treating infections caused by these organisms.

Anticryptococcal type D antibodies raised in rabbits.

Antibodies to Cryptococcus neoformans Type D have been raised in rabbits. The partially purified antibody pool was fractionated by affinity-chromatography, and it was shown that antibodies to the capsular polysaccharide of Type D were present in the pool. Neither methyl alpha-D-mannopyranoside, nor D-mannose were inhibitors of the binding of Type D polysaccharide to the antibody-pool. Thus it appears that the serum contains antibody populations which are specific for terminal side-chain carbohydrate moieties of the immunizing antigen.

Defining opportunistic invasive fungal infections in immunocompromised patients with cancer and hematopoietic stem cell transplants: an international consensus.

During the past several decades, there has been a steady increase in the frequency of opportunistic invasive fungal infections (IFIs) in immunocompromised patients. However, there is substantial controversy concerning optimal diagnostic criteria for these IFIs. Therefore, members of the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group formed a consensus committee to develop standard definitions for IFIs for clinical research. On the basis of a review of literature and an international consensus, a set of research-oriented definitions for the IFIs most often seen and studied in immunocompromised patients with cancer is proposed. Three levels of probability are proposed: "proven," "probable," and "possible." The definitions are intended for use in the context of clinical and/or epidemiological research, not for clinical decision making.

Cloning, sequencing, expression and allelic sequence diversity of ERG3 (C-5 sterol desaturase gene) in Candida albicans.

The C-5 sterol desaturase gene (ERG3), essential for yeast ergosterol biosynthesis, was cloned and sequenced from Candida albicans by homology with the Saccharomyces cerevisiae ERG3. The ERG3 ORF contained 1158bp and encoded 386 deduced amino acids. The clone was used to transform a gal1 mutant derived from the Darlington strain of C. albicans, using galactose selection. The Darlington strain is known to lack Delta(5,6) sterols, i.e. to have an erg3 phenotype (Howell, S.A., et al., 1990. J. Appl. Bacteriol. 69, 692-696). The transformant (CDTR1) contained six tandem integrated ERG3GAL1 repeats, had double the abundance of ERG3 transcript found in the host strain, and synthesized ergosterol, a Delta(5,6) sterol.The Darlington strain was noted to have an abundance of ERG3 transcript. Both ERG3 alleles in Darlington were cloned and sequenced in order to look for changes that might explain the erg3 phenotype. One allele, called Dar-2, contained a stop codon in place of tryptophan-292. The other ERG3 allele, called Dar-1, had changes in three amino acids, two of which were conserved in three fungal and one plant species. EcoRI genomic fragments containing ERG3 from the Dar-1 allele and from B311, the wild-type strain, were inserted into the plasmid pRS316 and used to transform a Saccharomyces cerevisiae erg3,ura3 mutant using uracil selection. The 4.1kb ERG3 fragments from the B311 and Dar-1 both contained 1. 4kb 5' and 1.5kb 3' flanking sequences around the coding region. Transformants with ERG3 from B311 but not from Dar-1 showed restored ergosterol synthesis. One or more of these three deduced amino acids in the Dar-1 allele of ERG3 appeared critical for function.

Chronic fungal sinusitis in apparently normal hosts.

Several fungal species are capable of causing either noninvasive fungal sinusitis or invasive disease characterized by erosion into mucosa, submucosa, bone, and deeper contiguous structures. The diagnosis of invasive infection becomes firmly established only after histologic demonstration of hyphae within these areas. Computerized tomography and magnetic resonance imaging can assist in distinguishing between invasive and noninvasive disease by outlining bone and adjacent structures. The 2 forms of chronic fungal sinusitis mandate different therapeutic approaches. While patients with noninvasive infection require only surgical removal of hyphal masses and the reestablishment of sinus drainage for a successful outcome, invasive infection necessitates not only thorough surgical debridement of abnormal tissues but may also require prolonged antifungal chemotherapy. All patients require long-term follow-up. Even the combined approach has sometimes proven disappointing during long-term follow-up of disease, rendering investigational therapy appropriate in some patients.

Catastrophic visual loss due to Cryptococcus neoformans meningitis.

We have reviewed our experience with 17 of our own patients with cryptococcal meningitis and 32 cases from the literature. Although this complication is an uncommon event, patients with cryptococcal meningitis may develop visual loss in the absence of other ocular lesions (endophthalmitis or cryptococcomas in the visual pathway) that could explain the visual symptoms. There are 2 distinct patterns of visual loss: rapid visual loss and slow visual loss. Rapid visual loss is characterized by onset of profound visual loss over a period as short as 12 hours before or early in the course of therapy and a clinical syndrome that is strongly suggestive of optic neuritis. Direct invasion of the optic nerve by C. neoformans is demonstrated by cases in this and other reports. Slow visual loss is characterized by slow but progressive visual loss which typically begins later during therapy and may be due to the effects of increased intracranial pressure. While the initial deficit may be mild, patients with slow visual loss can progress to severe visual loss over weeks to months. The only factors that appear to predict either pattern of visual loss are the presence of papilledema, an elevated CSF opening pressure, and a positive CSF India ink preparation. In the 25 visual loss patients for whom data were available for all 3 items, 10 (40%) were positive for all 3, as opposed to only 4 of 114 (3.5%) from a reference group of cryptococcal meningitis patients without visual loss (p < 0.00001). The only therapeutic measures with any degree of consistent success were those directed at reducing intracranial pressure. When begun early and used aggressively, such therapy halted and sometimes even reversed the course of visual loss, particularly in the slow visual loss group. Corticosteroids did not appear to be of value in the small number of patients who received them.

Minimal deviation melanoma with epidermotropic metastases arising in a congenital nevus.

A congenital nevus removed from a 20-year-old woman was found to contain a minimum deviation melanoma. Subsequently, there were multiple nodular recurrences in the region of the excision as well as in the grafted skin. Microscopically the recurrent lesions closely resembled the original nevus, including the junctional activity and even the grafted skin. Lymph node metastasis occurred 5 1/2 years after excision of the nevus. This is the first report of a minimal deviation melanoma arising in a congenital nevus with epidermotropic metastases.

Dental health and viridans streptococcal bacteremia in allogeneic hematopoietic stem cell transplant recipients.

Viridans streptococci were the most common cause of bacteremia in 61 consecutive myeloablative allogeneic hematopoietic stem cell transplant (HSCT) recipients, occurring in 19 of 31 bacteremic patients (61%) during the period of post-transplant neutropenia. Seven of the 19 had more than one viridans streptococcus in the same blood culture. Twenty isolates from 15 patients were Streptococcus mitis. Most viridans streptococci were resistant to norfloxacin, used routinely for prophylaxis. Comparison of the 19 patients with viridans streptococcal bacteremia with a contemporaneous group of 23 allogeneic HSCT recipients with fever and neutropenia but no identified focus of infection found that patients with viridans streptococcal bacteremia were more likely to have severe intraoral pathology while neutropenic (26% vs 0%) and slightly shorter interval between the last dental procedure and the onset of neutropenia (11 vs 14 days). Poor underlying dental health and the use of norfloxacin thus appear to predispose to viridans streptococcal bacteremia.

Assessment of new radioimmunoassay kit for determining urinary albumin at low concentrations: comparison with radial immunodiffusion.

The assay characteristics of a new radioimmunoassay kit for determining urinary albumin at low concentrations were studied. The sensitivity for urinary albumin was 2 mg/l, the analytical range 2 to 40 mg/l, and interassay coefficient of variation less than 12%. In a method comparison study entailing diabetic urine samples covering an albumin concentration of 2 to 150 mg/l the kit compared adequately with radial immunodiffusion (mean difference between methods = 2 mg/l; residual standard deviation = 4.6 mg/l), absolute variation between methods increasing with the concentration. The kit required much less skill than radial immunodiffusion but its capital and running cost were higher.

Parents' utilities for outcomes of occult bacteremia.

OBJECTIVE: To describe parents' values for outcomes of occult bacteremia using utility assessment, a quantitative method that incorporates risk preference. DESIGN: Computer-based utility assessment interview. SETTING: Urban children's hospital pediatric emergency department with 50 000 visits annually. PARTICIPANTS: Convenience sample of parents presenting with a child between 3 and 36 months. MAIN OUTCOME MEASURE: Parents' utility values for 8 outcomes from treatment of occult bacteremia: blood drawing, localized infection, hospitalization for antibiotics, meningitis with recovery, meningitis resulting in deafness, minor brain damage, severe brain damage, and death. RESULTS: Ninety-four subjects successfully completed the interview. Mean utilities were 0.9974 for blood drawing, 0.9941 for local infection, 0.9921 for hospitalization, 0.9768 for meningitis with recovery, 0.8611 for deafness, 0.7393 for minor brain damage, 0.3903 for severe brain damage, and 0.0177 for death. All values were significantly different from those that immediately preceded and succeeded (P<.0001), except for local infection vs hospitalization (P = .14). Median utilities for blood drawn, local infection, and hospitalization were 1. There were no significant differences among utilities of parents who presented with a febrile child (temperature > or =39 degrees C), or an afebrile child (temperature <39 degrees C). There were also no significant differences among utilities regardless of whether parents had children with prior experience with the outcomes. CONCLUSIONS: Assessment of utilities for outcomes of occult bacteremia yielded extremely high mean and median values for outcomes without permanent sequelae. This suggests that parents presenting to an emergency department may rationally prefer painful transient experiences, including venipuncture, for their children rather than risk even rare chances of severe outcomes.

Group B streptococcal necrotizing fasciitis arising from an episiotomy.

A case of extensive necrotizing fasciitis arising from an episiotomy is presented. Group B beta-hemolytic streptococcus and Staphylococcus aureus were isolated. Prompt recognition and aggressive therapy resulted in a favorable outcome despite significant morbidity. Salient clinical features of this rare postpartum complication are discussed and previous cases are reviewed. In addition to wide surgical resection, therapy included aggressive volume resuscitation with Swan-Ganz catheter monitoring, the use of military antishock trousers (MAST suit) to control diffuse hemorrhage, and temporary application of porcine xenografts.

Ultrasound evaluation of microanastomosis.

The Doppler ultrasound flowmeter with a pencil probe, emitting at a frequency of 10 megacycles/second, was used to making recordings along intact and repaired digital arteries and veins. In normal digital arteries, the flow averaged 3.78 ml/min. The ability of the method to appraise the condition of an anastamosis was evaluated in five patients with partial or complete digit, hand, or arm replants, and found quite satisfactory. Beginnings were made in developing criteria for satisfactory flow in these small arteries.

A ganglion of the temporomandibular joint presenting as a parotid tumor.

A case of temporomandibular joint (TMJ) ganglion is described, and 4 previously reported cases are noted. In all cases, initial diagnosis of parotid neoplasm proved incorrect at surgical exploration. Management of all cases consisted of identification of the facial nerve and excision of the ganglion with a cuff of TMJ capsule. The diagnosis of TMJ ganglion should be entertained when a presumed parotid neoplasm changes in size or position with jaw movement.

Skin and soft tissue injuries of the hand in children.

Three pediatric hand injury subtypes and their management have been presented. Finger tip injuries, while often minor and self-healing in children, may require surgical repair if the nailbed is disrupted or if there has been significant skin or pad loss from the tip. The decision between local repair and skin graft or pedicle reconstruction depends on the nature and extent of the injury. Multiple digit injuries should be carefully evaluated and manged with a view toward retention of digit length and restoration of function. Immediate closure of all wounds, while desirable, is not necessary. Minor hand burns are quite easily managed but burn injuries more severe in extent and/or depth must be treated with careful attention to detail. Splinting and elevation of the hand, early incisional decompression, the institution of early and diligent active motion and expeditious wound closure with skin grafts where full thickness loss has occurred will hasten the time of healing, minimize deformity and assist in the recovery of normal hand function.

Reconstruction of lateral nasal defects.

In planning reconstruction of a lateral nasal soft tissue defect, the surgeon should be aware of alternative methods and select the one most appropriate for the patient. Variables to be considered include the extent, depth, and location of the defect, the age, sex, and tissue laxity of the patient, and the availability of donor tissue. Surface defects are often best corrected with full-thickness skin grafts: upper eyelid, preauricular, postauricular, or supraclavicular, depending on which portion of the nose is being treated. Anterior superior helix composite grafts serve quite nicely to restore full-thickness alar losses, and other such defects are better reconstructed with a nasolabial flap. The midline forehead flap is quite useful but in certain situations has drawbacks. For larger skin and soft tissue defects, a transverse forehead flap based on the superficial temporal vessel provides appropriate reconstruction.

Cancer of the nose: ablation and repair.

Cancer of the nose is very common and lesions seen by reconstructive surgeons are often recurrent and extensive. Surgical removal of cancer of the nasal skin can usually be accomplished under local anesthesia, and in most instances frozen section histologic examination should be used to confirm the adequacy of excision. The location and three dimensional extent of the tumor will dictate the choices of repair or reconstruction as well as the timing thereof. Very small lesions can be excised with primary closure; other well circumscribed tumors can be excised and the defect closed with an appropriate nasal flap. In our experience most nasal skin cancers have been managed by excision and full-thickness skin grafting. We have found the skin of the neck and that of the preauricular region to provide the best skin cover except in the upper third of the nose where upper eyelid skin provides excellent coverage. We have used composite grafts from the ear to replace up to two-thirds of an alar rim. Nasolabial, cheek, and midline forehead flaps are useful in a variety of instances, but usually when less than one-half of the nose has been excised. We have been pleased, in most instances, with the Converse scalping flap for near total, subtotal, and extensive three-dimensional lower nasal defects. (Transverse superficial temporal artery pedicle flaps can be successfully used to reconstruct large nasal defects with adjacent cheek loss.) We have rarely used distant flaps. Regardless of what regional pedicle flap has been transferred to the nose, subsequent revisions of a relatively minor nature will nearly always enhance the result. Patients who have undergone extended total nasectomies are probably best managed with a prosthesis, as prognosis is often guarded and flap reconstruction may be quite unsatisfactory. In our experience, defects in lining and support can usually be repaired with local nasal tissue.