Late and atypical cytomegalovirus disease in solid-organ transplant recipients.

Posttransplantation cytomegalovirus (CMV) disease typically occurs 1-4 months after solid-organ transplantation. The case definition invariably includes unexplained fever for > or =3 days, often with leukopenia. Late and atypical presentation of CMV disease has been rarely reported. Five cases of late and atypical CMV disease in heart (n = 1), liver (n = 1), and kidney (n = 3) transplant recipients occurred within a 4-month period in early 1999. These patients presented at a median of 25 months after organ transplantation (range, 6 months to 22 years). Atypical findings included absence of fever in 3 patients, elevated white blood cell counts in 4 patients, and normal platelet counts in 4 patients. Four patients were at risk for primary CMV infection, and 3 received ganciclovir prophylaxis for 3 months. One patients was treated for rejection, and 2 patients had induction muromonab-CD3 (Orthclone; Orthobiotech). Two of the patients had pulmonary CMV disease, but neither of these patients had hypoxia. Two patients had enterocolitis, one of whom had chronic colitis for a year. These cases may represent a changing epidemiology and clinical presentation of CMV disease in solid-organ transplant recipients in an era of changing immunosuppression and improved CMV disease prevention in the early posttransplantation period.

Tween 80 opacity test responses of various Candida species.

A total of 111 Candida isolates representing 11 species were examined for their respective responses to a Tween 80 opacity test. The strains of Candida albicans and C. tropicalis that were examined produced an opacity response around their colonies at 2 to 3 days postinoculation. A second group of Candida species yielded a halo around their colonies at 8 to 10 days postinoculation. The remaining Candida species did not produce a positive test response through 10 days postinoculation. The strains of C. dubliniensis were easily differentiated from strains of C. albicans by this test. The Tween 80 opacity test is simple and economical to prepare and is easy to interpret.

Evaluation of a water-extracted pharmamedia agar medium for the culture of fungi.

The purpose of this investigation was to determine the value of a water-extracted Pharmamedia agar medium for the culture of fungi. The growth of the fungi on this medium was compared to that on Sabouraud brain heart infusion agar. The Pharmamedia extract agar supported the growth of the 42 filamentous and yeast or yeast-like fungi that were examined. The medium was relatively clear to easily permit observation of the diagnostic microscopic and macroscopic architecture of the organisms. The Pharmamedia extract agar also permitted the conversion of the mold-to-yeast phase with Blastomyces dermatitides and Penicillium marneffei. This investigation supports the use of Pharmamedia as a culture medium for the clinical microbiology laboratory. Its relative simplicity of preparation and effective use as a culture medium, as well as a conversion medium, emphasizes its useful application for clinical mycology.

U.S. case report of cerebral phaeohyphomycosis caused by Ramichloridium obovoideum (R. mackenziei): criteria for identification, therapy, and review of other known dematiaceous neurotropic taxa.

We report a case of cerebral phaeohyphomycosis in a 36-year-old male caused by the neurotropic fungus Ramichloridium obovoideum (Matushima) de Hoog 1977 (Ramichloridium mackenziei Campbell et Al-Hedaithy 1993). This man resided in the Middle East, where the fungus appears to be endemic and, possibly, geographically restricted, since all previous reports of brain abscesses due to this organism have been for patients indigenous to this area. As a servant of the Saudi Arabian royal family, he appeared in the United States seeking treatment for chronic weight loss, fatigue, decreased memory, and a more recent 2-week history of right-hand weakness which worsened to involve the entire right upper extremity. On the day prior to his admission, he had a focal motor seizure with rotation of the head and eyes to the right, followed by secondary generalization. A computerized tomogram showed a ring-enhancing hypodense lesion in the left parietal subcortical region with associated edema and mass effect. Diagnosis of a fungal etiology was made following a parietal craniotomy and excisional biopsy by observation of septate, dematiaceous hyphal elements 2 to 3 microm in width on hematoxylin-and-eosin-stained sections from within areas of inflammation and necrosis. Culture of the excised material grew out a dematiaceous mould which was subsequently identified as R. obovoideum. At two months postsurgery and with a regimen of 200 mg of itraconazole twice a day, the patient was doing well and returned to Saudi Arabia. His condition subsequently deteriorated, however, and following a 7-month course of itraconzole, he expired. We use this case to alert clinicians and personnel in clinical mycology laboratories of the pathogenicity of this organism and its potential occurrence in patients with central nervous system signs and symptoms who have resided in the Middle East and to review and/or compare R. obovoideum with other neurotropic, dematiaceous taxa and similar nonneurotropic, dematiaceous species.

Comparison of the Histopaque-1119 method with the Plasmagel method for separation of blood leukocytes for cytomegalovirus isolation.

Histopaque-1119 (Sigma Chemical Co., St. Louis, Mo.) and Plasmagel (Cellular Products, Inc., Buffalo, N.Y.) were compared as density gradient separation reagents for the separation of polymorphonuclear leukocytes and mononuclear cells from blood from the isolation of cytomegalovirus (CMV). Of 200 peripheral blood specimens examined, CMV was recovered from 51 by both methods. The time of detection of immunofluorescent sites or a cytopathic effect associated with CMV was similar by each method. The Histopaque-1119 method was less time-consuming than the Plasmagel method since it did not require a precentrifugation step for the settling of erythrocytes. The use of Histopaque-1119 will permit an effective alternative single-step method for the separation of blood leukocytes for the isolation of CMV.

Production and characterization of improved liposomes containing radiographic contrast media.

The authors have investigated liposomes prepared by the reverse-phase evaporation method (REVs) and a modification of this technique that employs a microemulsifier (MREVs), for the delivery of radiographic contrast media (RCM) to the liver and spleen. The modification entailed substituting a Microfluidizer (Microfluidics Inc., Newton, MA) for the sonication step of the REV technique. The MREV procedure is amenable to large-scale production and continuous-flow operation and yields products with high RCM encapsulation. Efficiently entrapped are ionic, high-osmolar diatrizoate (24.38 +/- 2.62% versus 8.35 +/- 0.55%; MREV versus REV), and nonionic, low osmolar Iotrolan (Schering AG, Berlin, FRG) (24.84 +/- 2.13% versus 7.25 +/- 1.19%) RCM with iodine-to-lipid ratio of 1.5:1. The MREV procedure, therefore, has practical advantages over the REV method. High liver and spleen uptakes of Iotrolan-containing vesicles were noted in normal rats. The diatrizoate MREVs lost their contents on contact with serum, resulting in urinary excretion of this agent. Computed tomography values of splenic and hepatic sections, 1 hour after intravenous injection of Iotrolan MREV (500 mgI/kg), are 0.78, and 0.08 Hounsfield Units (HU)/mgI/kg, respectively (versus 0.01, and 0.006 HU/mgI/kg for free Iotrolan).

Biodistribution and clearance of contrast-carrying MREV liposomes.

Contrast-carrying liposomes (CCLs) have been shown to increase the attenuation coefficient of the liver and spleen during computed tomographic (CT) imaging. A modification of the reverse-phase evaporation preparative technique ("MREV") led to production of CCLs that entrap contrast media efficiently. After intravenous injection, MREVs are phagocytized by the Kupffer cells of the liver and the macrophages of the spleen. The biodistribution and clearance of MREVs were studied to evaluate their potential for clinical use, MREVs carrying iodine-125-iotrolan were administered intravenously to 12 rats at doses of 400 mg lipid (containing 400 mg iodine) per kilogram body weight. Pairs of rats were anesthetized and scanned at 3 hours, and 1, 7, 14, 27 and 48 days; CT attenuation values of liver, spleen, blood, kidneys and bladder were measured. Immediately following CT, the rats were killed and tissue specimens were radioassayed. Maximum iodine content in liver and spleen was reached at one day and sustained at high levels for seven days. Biologic removal half-time of the agent in both the liver and spleen was six days. Maximum CT enhancements over baseline were observed at 24 hours and reached 210 delta HU in the liver and 880 delta HU in the spleen per gram iodine injected per kilogram body weight. It is concluded that MREVs have appropriate imaging characteristics, biodistribution, and clearance to be effective CT contrast agents.

Lectins and their application to clinical microbiology.

Lectins are generally associated with plant or animal components, selectively bind carbohydrates, and interact with procaryotic and eucaryotic cells. Lectins have various specificities that are associated with their ability to interact with acetylaminocarbohydrates, aminocarbohydrates, sialic acids, hexoses, pentoses, and as other carbohydrates. Microbial surfaces generally contain many of the sugar residues that react with lectins. Lectins are presently used in the clinical laboratory to type blood cells and are used in a wide spectrum of applications, including, in part, as carriers of chemotherapeutic agents, as mitogens, for fractionation of animal cells, and for investigations of cellular surfaces. Numerous studies have shown that lectins can be used to identify rapidly certain microorganisms isolated from a clinical specimen or directly in a clinical specimen. Lectins have been demonstrated to be important diagnostic reagents in the major realms of clinical microbiology. Thus, they have been applied in bacteriology, mycology, mycobacteriology, and virology for the identification and/or differentiation of various microorganisms. Lectins have been used successfully as epidemiologic as well as taxonomic markers of specific microorganisms. Lectins provide the clinical microbiologist with cost-effective and potential diagnostic reagents. This review describes the applications of lectins in clinical microbiology.

Rapid detection of herpes simplex virus with fluorescein-labeled Helix pomatia lectin.

The use of fluorescein-conjugated Helix pomatia lectin was shown to be as effective as fluorescein-conjugated monoclonal antibody reagents for the detection and differentiation of herpes simplex virus type 1 and type 2 (HSV-1 and HSV-2) in MRC-5 cell culture. Cells infected with HSV-1 generally displayed a pattern of nongranular or diffuse fluorescence, while cells infected with HSV-2 were identified by the production of fluorescent grains and flecks. This unique nonimmunological reagent, when used in combination with low-speed centrifugation, provides a remarkably specific, sensitive, rapid, and cost-effective means to detect HSV-infected MRC-5 or BHK-21 cells as early as 20 h postinoculation. In contrast to the immunofluorescence method, the serotypes of HSV can be differentiated with only one fluorescein-H. pomatia reagent in MRC-5 cell cultures.

Light-induced conductivity changes of purple membrane suspensions in strong electrolytes.

Measurements have been made of the modulated light-induced changes in conductivity and the associated relaxation times of bacteriorhodopsin in a variety of strong electrolytes, both unbuffered and buffered. The effects of pH and temperature variation have been studied as well as the effect of adding valinomycin. Two relaxation times can be distinguished: a fast lifetime associated with protonation-deprotonation, and a slow lifetime associated with ion binding. The ion-binding effects appear to be cation specific.

Congo red as a fluorochrome for the rapid detection of fungi.

Congo red may be applied as a fluorochrome to rapidly detect fungi in clinical specimens, tissue, and fungal culture preparations. This generally available stain is cost effective and simple to prepare. The stain may be prepared with potassium permanganate as a counterstain or with Formalin or glutaraldehyde as a fungicide.

Immunodiagnosis of adult chlamydial conjunctivitis.

This study presents data from a prospective comparison of four currently available diagnostic tests for Chlamydia trachomatis infection. Seventy-six patients clinically suspicious for chlamydial conjunctivitis were all tested with Giemsa stain cytology, direct monoclonal fluorescent antibody (DFA) microscopy, enzyme immunosorbent assay (EIA) for chlamydial antigens, and standard McCoy cell culture. When compared with primary cell culture, diagnostic Giemsa inclusions had a sensitivity and specificity of 43 and 100%, respectively, supportive Giemsa cytology 71 and 67%, the enzyme immunoassay 71 and 97%, and the monoclonal fluorescent antibody 57 and 81%. Each nonculture method has distinct advantages in terms of cost, technical difficulty, speed, and accuracy, which dictate selection of the most appropriate test for office or laboratory diagnosis of chlamydial conjunctivitis.

Stool caproic acid for screening of Clostridium difficile.

Clostridium difficile is the prime etiologic agent in the production of pseudomembranous colitis by its powerful cytotoxin. The most common test for the toxin is a tissue culture method with neutralization of cytopathic effect by a C. difficile antiserum. This method is expensive and requires a minimum of 72 hours before results can be obtained. Attempts to create a rapid method, counterimmunoelectrophoresis, enzyme-linked immunosorbent, latex agglutination, and fluorescent antibody test are fraught with many problems. This report describes a rapid method for the identification of C. difficile, using gas-liquid chromatography (GLC) for the demonstration of caproic acid, a product of the organisms fatty acid metabolism.

Serogrouping single colonies of beta-hemolytic streptococci with achromopeptidase extraction.

Achromopeptidase (TBL-1; Wako Chemicals, USA, Inc., Dallas, Tex.) prepared as a 2,000-U/ml solution will extract the serogroup antigens from single colonies of groups A, B, C, F, and G streptococci in 1 min at room temperature. This enzyme extraction is not effective for the serogrouping of all group D streptococcus species. Achromopeptidase extracts can be used with latex or coagglutination reagents.

Identification of group B streptococcal antigen with lectin-bound polystyrene particles.

The lectin of the tomato, Lycopersicon esculentum, or of the potato, Solanum tuberosum, can be passively coupled to amide-modified polystyrene spheres to be used as a detection reagent for the specific identification of group B streptococcal cultures grown in selective or nonselective Todd-Hewitt broth for 5 and 4 h, respectively. Agglutination occurred when the lectin reagents were allowed to react with either the cell suspension, clarified broth, or antigen extracts from group B streptococci grown in Todd-Hewitt broth. No agglutination occurred when these lectins were allowed to react with strains of serogroup A, C, D, F, or G streptococci. False-negative agglutination responses may occur with certain serotype of group B streptococci grown on Columbia sheep blood agar. A 20-min staining time permitted the specific labeling of fixed smears of group B streptococci with fluorescein-conjugated Lycopersicon lectin. The lectin from the solanaceous plant Datura stramonium did not agglutinate group B streptococci or other clinically significant streptococcal serogroups.

Rapid identification of group JK and other corynebacteria with the Minitek system.

Forty primary clinical isolates and 50 stock cultures of corynebacteria and coryneform bacteria were tested with the Minitek system (BBL Microbiology Systems, Cockeysville, Md.). The Minitek correctly identified all of these organisms, including JK group isolates, within 12 to 18 h of incubation. The method does not require serum supplements for testing carbohydrate utilization by the bacteria. The Minitek system is an extremely simple and rapid way to identify the JK group, as well as many other corynebacteria, by established identification schemata for these bacteria.

Brain abscess due to Haemophilus aphrophilus: possible canine transmission.

A 58-year-old patient developed progressive right hemiparesis and a hemisensory loss. Computed tomography demonstrated a lesion in the left frontoparietal region with ring enhancement. A craniotomy was performed and an abscess was removed, which on culture grew Haemophilus aphrophilus. The same organism was isolated from the patient's poodle dog but not from three other poodles of family members. This, along with previous reports, suggests that the poodle may be a vector in the transmission of this organism, which rarely may cause a brain abscess.

Photoacoustic spectroscopy of purple membrane fragments. Evidence for disorder during photocycle.

A strong band at 412 nm has been observed in the photoacoustic spectrum of partially dried purple membrane, peaking sharply at a modulation frequency of about 70 Hz. This may be explained in terms of a disorder-order transition.

Photo-oxidation of tetracycline adsorbed on hydroxyapatite in relation to the light-induced staining of teeth.

In an attempt to understand the chemistry of the light-induced staining of children's teeth by tetracycline, we studied the photo-chemical behavior of tetracycline adsorbed on hydroxyapatite, as a simple model of enamel. Tetracycline was strongly bound by hydroxyapatite to give a pale yellow material which, under ultraviolet light, showed a bright yellow fluorescence (lambda max 525 nm). On exposure of this material to the radiation from a medium-pressure mercury lamp, the fluorescence gradually disappeared, and a red-purple product was formed. Photo-acoustic spectroscopy was employed to follow the disappearance of tetracycline and the concomitant formation of the red-purple product, the spectrum of which (lambda max 530 nm) led to the conclusion that it was 4 alpha, 12 alpha-anhydro-4-oxo-4-dedimethylaminotetracycline (AODTC). This assignment was supported by the observation that 1 mole of oxygen was absorbed per mole of adsorbed tetracycline converted to the red-purple product. It is suggested that the formation of AODTC on hydroxyapatite probably takes place by a mechanism of photo-oxidation similar to that already proposed for solutions of tetracycline, and that the formation of AODTC in children's teeth is responsible for the light-induced staining caused by tetracycline.