De novo donor specific antibodies and patient outcomes in renal transplantation.

Single antigen identification of HLA antibodies is used to detect donor specific antibodies (DSAs). However, the impact of DSA elements such as class, relative strength, duration, and longitudinal effect on graft function and survival, remains unclear. Routine DSAs (LabScreen, One Lambda, Inc., Canoga Park, CA) and metabolic studies were performed at 1, 3, 6, 9, and 12 months post-transplant, and every 6 months for renal transplant recipients from 7/2007-7/2010 (n = 389). Biopsies were evaluated by updated Banff 2005 guidelines after two consecutive positive DSAs. Based on these tests, 25% of recipients developed de novo DSA. Those with DSA had increased acute rejection episodes (AR), higher creatinine (Scr), and worse graft survival. Three subgroups of these patients were identified based on duration: persistent DSA (> 1), isolated DSA, or no DSA. Persistent DSA patients were more likely to be African American, and have higher mean fluorescence intensity (MFI) and AR rates. Persistent DSA patients, with or without AR, had elevated Scr. Recipients with DQ-only DSA had higher rates of antibody mediated rejection (AMR). From this, we conclude that routine posttransplant DSA monitoring identifies recipients at risk for graft damage or loss. Persistent de novo DSAs correlated with inferior graft outcomes and AMR. With or without AR, DSA persistence was associated with worse outcomes, possibly warranting intervention. De novo DQ-DSA may be a biomarker for chronic damage and/or AMR, while an isolated DSA determination appears clinically insignificant.

Performance assessment of the fecal leukocyte test for inpatients.

Traditionally, fecal leukocyte testing detects large bowel inflammation or disruption, conditions that allow leukocytes into the stool. However, test usefulness with inpatients is unclear. Two hundred five inpatients who had undergone one to three tests were identified, and their FLT results were compared to their gastrointestinal disease diagnoses at time of discharge. A specificity of 92% for detecting intact colonic mucosae in inpatients was found.

Comparison of panel-reactive antibody levels in Caucasian and African American renal transplant candidates.

PRA levels from 58 Caucasian and 70 African American ESRD patients were compared against a panel of cryopreserved lymphocytes from 60 donors (40 Caucasian, 15 African American, 5 others) to determine whether there was significant racial influence on PRA outcome. African Americans were found to have significantly higher mean PRA levels than Caucasians (27% vs. 18%, P = 0.02). Restricting this analysis to only 1 degree transplant candidates showed predictably lower mean PRAs: 6% in Caucasians and 15% in African Americans, but the difference between the two groups remained significant (P = 0.015). The percentage of patients with PRA > or = 10% was also greater among African Americans than Caucasians (43% vs. 24%, P = 0.026). For patients not previously transplanted, the difference between these frequencies remained significant: 11% in Caucasians, 30% in African Americans (P = 0.025). Untransplanted African American patients with positive PRAs (> or = 10%) had significantly higher PRA against African American cell donors (mean = 55%) than against Caucasian cell donors (mean = 44%) (mean difference = 10.6%, P = 0.0056). African Americans were more frequently transfused than Caucasians. The percentage of patients not previously transplanted receiving 0, 1-5, and > 5 transfusions were 69%, 22%, and 9% for Caucasians and 43%, 44%, and 13% for African Americans (P = 0.03). This higher transfusion rate is the most likely contributor to the elevated PRA levels observed in African Americans.

Evaluation of the Baxter-MicroScan 4-hour enzyme-based yeast identification system.

A new 4-h Yeast Identification Panel (YIP; Baxter-MicroScan, W. Sacramento, Calif.) was compared with the API 20C Yeast Identification System (Analytab Products, Inc., Plainview, N.Y.) in the identification of recent clinical yeast isolates. The YIP had a 94% correlation (288 of 306) in identifying 22 species within the genera Candida, Hansenula, Pichia, Rhodotorula, Saccharomyces, and Torulopsis. Correlation dropped to 65% for those species within the genera of slower growing yeasts, i.e., Blastoschizomyces spp., Crpytococcus spp., Geotrichum spp., Hyphopichia spp., Phaeococcomyces spp., Prototheca spp., and Trichosporon spp. Overall correlation with the API 20C was 92% (365 of 401) for those taxa included in the data base and 85% (373 of 437) for all yeasts encountered in the study. There were 36 (8.2%) discrepant identifications, which were due in part to the limited data base. Expansion of the data base plus the easy inoculation, reading, and rapid results of the YIP should make it an excellent method for yeast identification.

Evaluation of the updated Vitek yeast identification data base.

Using 398 isolates of yeasts and yeastlike fungi comprising 9 genera and 26 species, as well as the hyphomycete Geotrichum candidum and the achlorophyllous alga Prototheca wickerhamii, we compared the API 20C yeast identification system with the modified Vitek yeast identification system with an expanded data base. We found 11 discrepancies between the two systems: five (1.3%) of the isolates (Blastoschizomyces capitatus, 1; Candida albicans, 1; Hansenula anomala, 1; Rhodotorula minuta, 2) had biocodes not included in the expanded Vitek data base, and six (1.5%) of the isolates (Candida lusitaniae, 1; Candida parapsilosis, 1; Cryptococcus uniguttulatus, 1; H. anomala, 1; Torulopsis candida, 2) were misidentified by the Vitek system. Overall, the efficacy of the Vitek system compares favorably with that of the API 20C in the identification of clinically important yeasts.

Uncommon yeastlike zoopathogens and commercial systems for their identification.

Opportunistic infections caused by yeasts or yeastlike fungi have increased in incidence in recent years as a result of clinical and therapeutic factors. Several formerly uncommon yeastlike zoopathogens--Candida lusitaniae, Candida paratropicalis (sucrose-negative variant of Candida tropicalis), Trichosporon beigelii, Blastoschizomyces capitatus, and Rhodotorula species--have been isolated from patients with invasive infections. The increased isolation of such opportunistic pathogens from a variety of clinical specimens has created a demand for simple, rapid, reliable, and accurate commercial systems to assist laboratorians in identification. Here we summarize the manual and automated systems currently available and present detailed descriptions of three representative commercial products, i.e., API 20C, Abbott Quantum II, and API Yeast-Ident.

Evaluation of YeastIdent and Uni-Yeast-Tek yeast identification systems.

The accuracy of the new API YeastIdent system and the Flow Laboratories Uni-Yeast-Tek identification kit with an expanded data base was evaluated in comparison to the API 20C yeast identification system by three laboratories. A total of 489 test isolates were used, biased toward yeasts commonly encountered in clinical specimens. Isolates not in a system's data base were not counted in the evaluation of that system. For isolates in their data base, YeastIdent was 55% accurate and Uni-Yeast-Tek was 40% accurate. By the manufacturer's criteria of reliable identification without additional tests, both systems failed to identify many common and uncommon species. The limited number of substrates and difficulties in assessing results obtained with 11 of the API YeastIdent substrates and apparent errors in the expanded Uni-Yeast-Tek data base appeared to be major factors limiting the accuracy of these systems.

Taxonomic and nomenclatural evaluation of the genera Candida and Torulopsis.

It is concluded that the validly published genera Candida and Torulopsis are taxonomically distinct. The recently proposed merger of these two yeast genera is rejected.

Evaluation of the exoantigen test for identification of Histoplasma species and Coccidioides immitis cultures.

Recently it was demonstrated that certain fungal pathogens could be rapidly and accurately identified on the basis of their exoantigens. Mycelial-form cultures of the Histoplasma species consistently appear to produce either H or M antigens, or both, whereas cultures of Coccidioides immitis produce tube precipitinogen, heat-labile precipitinogen, or heat-labile F antigens. Three laboratories each evaluated 48 cultures by the exoantigen procedure for the identification of Histoplasma spp. and C. immitis. One hundred forty-four cultures were tested, and 143 (99.3%) were correctly identified as Histoplama spp. or C. immitis, or neither. The simple and rapid exoantigen test appears to be a sensitive and specific method for identification of these fungi.

An ultrastructural comparison of the cell envelopes of selected strains of Nocardia asteroides and Nocardia brasiliensis.

Growth curves were determined for three strains each of Nocardia asteroides and Nocardia brasiliensis. Two strains of N. brasiliensis and one strain of N. asteroides had longer lag periods of growth than the remaining three strains. All strains had generation times of approximately 5.5 hours. The ultrastructure of the cell envelope of each Nocardia strain in early stationary phase growth was also examined. All the strains had typical trilaminar cell walls and cell membranes. The thickness of the cell wall layers, especially the inner peptidoglycan layer, varied from strain to strain. The inner layer of two strains of N. brasiliensis and one strain of N. asteroides was 12 nm or more in thickness, while that of the remaining three strains was 7 nm thick. These observed differences in growth patterns and/or thickness of the cell wall layers could be correlated to the varying degrees of virulence as well as the divergent pathologies exhibited by these organisms.

Assimilation of protocatechuic acid and p-hydroxybenzoic acid as an aid to laboratory identification of Candida parapsilosis and other medically important yeasts.

Test for the ability of yeasts isolated from clinical specimens to utilize protocatechuic acid and p-hydroxybenzoic acid were carried out by using techniques that are commonly employed to test assimilation of carbon sources. A total of 60 isolates of Candida parapsilosis and 5 isolates of Candida humicola readily assimilated these two phenolic acids, whereas other Candida species gave uniformly negative results. Cryptococcus albidus, Cryptococcus terreus, and some isolates of Cryptococcus laurentii also assimilated protocatechuate and p-hydroxybenzoate, whereas Cryptococcus neoformans did not. Results of these tests suggest that assimilation of protocatechuate and p-hydroxybenzoate may be a useful characteristic, when used in conjunction with traditional tests, for identifying C. parapsilosis and C. albidus.

Evaluation of the new API 20C strip for yeast identification against a conventional method.

The new API 20C yeast identification system together with appropriate microscopic morphology determinations achieved a 97% correlation with a rapid conventional method. Whereas a group composed of Candida, Torulopsis, Saccharomyces, and Rhodotorula was identified with ease (98% overall correlation), a second group, containing Cryptococcus, Trichosporon, and Geotrichum species, appeared to give the system the most difficulty (90% correlation). Within this group particular difficulty was encountered in identifying varieties of Cryptococcus albidus, C. terreus, C. laurentii, Trichosporon beigelli, and Geotrichum spp. as to species. The API 20C system should be incubated the full 72 h prescribed by the manufacturer. However, when used in conjunction with appropriate morphological tests, presumptive identifications of some Candida and Torulopsis species may be made at 24 to 48 h. To facilitate identifications of the more difficult group of yeasts, ancillary tests for determining nitrate reductase, urease, and phenol oxidase activities should be considered as additions to the strip. Incorporating the phenol oxidase test would be especially important for identification of Cryptococcus neoformans, a yeast which should be identified as quickly and as accurately as possible. The API 20C system with computer assistance has proved to be an easy-to-inoculate, versatile, and fairly rapid method of yeast identification, giving results comparable to those obtained by conventional methodologies.

Improved blood culture technique based on centrifugation: clinical evaluation.

A total of 3,335 blood samples from 1,180 patients suspected of having bacteremia were analyzed concurrently by two methods: (i) supplemented peptone broth with sodium polyanethanol sulfonate and a CO2 atmosphere; and (ii) lysis centrifugation at 3,000 X g for 30 min onto a high-density, hydrophobic cushion. The centrifugation technique recovered 80% of the positive cultures as compared with 67% for the broth method. The centrifugation technique showed an apparent increase in the isolation of staphylococcus aureus, Pseudomonas, and yeasts. In almost every instance, the time required for detection of a positive culture was shortest for the centrifugation method. Contamination rates for both systems were comparable (1.4%). Quantitation, offered only by the centrifugation method, proved useful on several occasions in discriminating between an opportunistic infection versus a skin contaminant and in judging efficacy of antimicrobial therapy.

Isolation and rapid identification of yeasts from compromised hosts.

In order to improve the isolation and identification of yeasts in a cancer research hospital, a protocol was developed utilizing an improved blood culture methodology and a four-test schema for rapid yeast identification. The blood culturing technique, based upon centrifugation, has shown a ten-fold increase in isolation of fungi from blood and has provided for: quantitation or organisms, unlimited selection of media and atmospheres for primary culturing, and a 1:200 dilution of microorganisms away from serum antimicrobial factors and antibiotics. The four-test schema, which may be adapted for the identification of any unknown yeast in pure culture, consists of a dye pour plate auxanogram (DPPA), Tween 80-Oxgall-Caffeic acid (TOC), a rapid nitrate-reductase test (swab test) and Urea 'R' Broth. Using this protocol, over 95% of the clinical isolates received were correctly identified within 24 hours and 100% by 48 hours. By using DPPA, a 14 sugar assimilation pattern for each isolate was determined within 12 to 16 hours; and in some cases, as little as 6 hours. Growth on TOC yielded one of the following results: (1) Candida albicans and Candida stellatoidea sequentially produced germ tubes and chlamydospores in 3 hours and 24 hours, respectively; (2) Cryptococcus neoformans produced a brown pigment specific for its identification in 12 hours or less. The swab test gave results on nitrate utilization in less than 15 minutes and urease was detected within 4 hours.

Immunodiagnosis of histoplasmosis in a compromised host.

Three serological tests for the diagnosis of histoplasmosis were compared for sensitivity and specificity in serum from blood bank donors, patients with histoplasmosis, and infected or noninfected immunosuppressed patients. The histoplasmin latex agglutination test was positive in 9% of the normal patients, 33% of the histoplasmosis patients, and 61% of the noninfected immunosuppressed patients. Since the test is prone to many false-positive results in patients with inflammatory diseases or non-Histoplasma infections, it has limited potential as a screening test among compromised patients. Immunodiffusion and counterimmunoelectrophoresis using a mycelial antigen were found to be more sensitive than either test using a combined yeast and mycelial antigen or a pure yeast phase antigen. Counterimmunoelectrophoresis at pH 7.2 proved to be the test of choice for serodiagnosis of histoplasmosis, resolving 85% of the immunocompetent infected patients and 100% of the infected immunosuppressed patients. Results indicated that counterimmunoelectrophoresis in conjunction with immunodiffusion could be used as a screening protocol to determine infection in incoming patients in a cancer hospital.

Rapid urea broth test for yeasts.

A rapid, miniaturized, urea broth test useful for detecting urease activity of yeasts was compared to Christensen urea agar. All urease-producing yeasts tested were positive on both media; however, 60% were reactive in the urea R broth within 30 min, and the remainder were reactive within 4 h. This urea multiwell test may be useful as a rapid screening method for detecting urease-producing yeasts recovered from clinical specimens and as an adjunct test with other rapid methods of yeast identification.

Rapid method for determining nitrate utilization by yeasts.

A test for the nitrate-reductase activity in yeasts has been developed, in which the reaction may be read after only 10 min of incubation. The rapidity of the test is due to the optimization of pH, substrate concentration, and temperature for the reaction.

New culture medium for the presumptive identificaion of Candida albicans and Cryptococcus neoformans.

A new medium composed of Tween 80, oxgall, caffeic acid, and Davis agar (TOC) that provides for the rapid presumptive identification of Candida albicans and Cryptococcus neoformans is described herein. C. albicans is differentiated from other yeasts by the sequential production of germ tubes and chlamydospores. In a comparison with cormeal agar control plates, there was an increase of chlamydospore-forming strains of C. albicans (97.1% versus 87.2%) and a decrease in the time required for chlamydospore formation (24 h versus 48 h). C. neoformans produced a brown pigment of TOC, which is specific for its identification, thus differentiating it from the other yeasts. A comparison of 24-h pigment production by C. neoformans on TOC with that of birdseed agar showed a dark, coffee brown color in the former cultures and a light brown color in the latter. The change in pigmentation of C. neoformans, as well as morphological changes in C. albicans, can be induced within 3 to 12 h and in not more than 24 h on the TOC medium.

Cellular localization of uridine 5'-diphosphate-N-acetyl-D-glucosamine oxidoreductase activity in Citrobacter freundii.

The uridine 5'-diphosphate-N-acetyl-d-glucosamine oxidoreductase activity of Citrobacter freundii ATCC 10053 was found to be located in the soluble cytoplasmic fraction of lysed spheroplasts.

Improved auxanographic method for yeast assimilations: a comparison with other approaches.

An improved pour-plate auxanographic method has been developed for determining the assimilation of 14 different carbohydrates by medically important yeasts. Reduction of a dye incorporated into the agar has been correlated with the growth and carbohydrate assimilation of the yeasts, allowing the speciation of many yeasts within 24 to 48 h. This technique has been found to compare more than favorably with existing yeast assimilation techniques, in terms of rapid identification, total cost, and technician time in preparing and inoculating the plates. The dye pour-plate auxanographic technique provides an easier-to-interpret, rapid, and reproducible method of mycological identification for small clinical laboratories.