Urine Antigen Detection as an Aid to Diagnose Invasive Aspergillosis.

Background: Establishing rapid diagnoses of invasive aspergillosis (IA) is a priority tests that detect galactomannan and ß-d-glucan are available, but are technically cumbersome and rely on invasive sampling (blood or bronchoalveolar lavage). Methods: We optimized a lateral flow dipstick assay using the galactofuranose-specific monoclonal antibody (mAb476), which recognizes urine antigens after Aspergillus fumigatus pulmonary infection in animals. Urine samples were obtained from a cohort of 78 subjects undergoing evaluation for suspected invasive fungal infections, and stored frozen until testing. Urine was processed by centrifugation through desalting columns and exposed to dipsticks. Reviewers blinded to clinical diagnoses graded results. Western blots were performed on urine samples from 2 subjects to characterize mAb476-reactive antigens. Results: Per-patient sensitivity and specificity for diagnosis of proven or probable IA in the overall cohort was 80% (95% confidence interval [CI], 61.4%-92.3%) and 92% (95% CI, 74%-99%), respectively. In the subgroup with cancer, sensitivity was 89.5% (95% CI, 66.7%-98.7%) and specificity was 90.9% (95% CI, 58.7%-99.8%); among all others, sensitivity and specificity were 63.6% (95% CI, 30.8%-89.1%) and 92.9% (95% CI, 66.1%-99.8%), respectively. Eliminating lung transplant recipients with airway disease increased sensitivity in the noncancer cohort (85.7% [95% CI, 42.1%-99.6%]). Semiquantitative urine assay results correlated with serum galactomannan indices. Western blots demonstrated mAb476-reactive antigens in urine from cases, ranging between 26 kDa and 35 kDa in size. Conclusions: Urine testing using mAb476 may be used as an aid to diagnose IA in high-risk patients.

Brief Report: Willingness to Accept HIV-Infected and Increased Infectious Risk Donor Organs Among Transplant Candidates Living With HIV.

BACKGROUND: HIV-infected (HIV+) donor to HIV+ recipient (HIV D+/R+) transplantation might improve access to transplantation for people living with HIV. However, it remains unknown whether transplant candidates living with HIV will accept the currently unknown risks of HIV D+/R+ transplantation. METHODS: We surveyed transplant candidates living with HIV from 9 US transplant centers regarding willingness to accept HIV+ donor organs. RESULTS: Among 116 participants, the median age was 55 years, 68% were men, and 78% were African American. Most were willing to accept HIV+ living donor organs (87%), HIV+ deceased donor organs (84%), and increased infectious risk donor organs (70%). Some (30%) were concerned about HIV superinfection; even among these respondents, 71% were willing to accept an HIV D+ organ. Respondents from centers that had already performed a transplant under an HIV D+/R+ transplantation research protocol were more willing to accept HIV+ deceased donor organs (89% vs. 71%, P = 0.04). Respondents who chose not to enroll in an HIV D+/R+ transplantation research protocol were less likely to believe that HIV D+/R+ transplantation was safe (45% vs. 77%, P = 0.02), and that HIV D+ organs would work similar to HIV D- organs (55% vs. 77%, P = 0.04), but more likely to believe they would receive an infection other than HIV from an HIV D+ organ (64% vs. 13%, P < 0.01). CONCLUSIONS: Willingness to accept HIV D+ organs among transplant candidates living with HIV does not seem to be a major barrier to HIV D+/R+ transplantation and may increase with growing HIV D+/R+ transplantation experience.

Phosphorylation of Saccharomyces cerevisiae choline kinase on Ser30 and Ser85 by protein kinase A regulates phosphatidylcholine synthesis by the CDP-choline pathway.

The Saccharomyces cerevisiae CKI-encoded choline kinase is phosphorylated on a serine residue and stimulated by protein kinase A. We examined the hypothesis that amino acids Ser(30) and Ser(85) contained in a protein kinase A sequence motif in choline kinase are target sites for protein kinase A. The synthetic peptides SQRRHSLTRQ (V(max)/K(m) = 10.8 microm(-1) nmol min(-1) mg(-1)) and GPRRASATDV (V(max)/K(m) = 0.15 microm(-1) nmol min(-1) mg(-1)) containing the protein kinase A motif for Ser(30) and Ser(85), respectively, within the choline kinase protein were substrates for protein kinase A. Choline kinase with Ser(30) to Ala (S30A) and Ser(85) to Ala (S85A) mutations were constructed alone and in combination by site-directed mutagenesis and expressed in a cki1Delta eki1Delta double mutant that lacks choline kinase activity. The mutant enzymes were expressed normally, but the specific activity of choline kinase in cells expressing the S30A, S85A, and S30A,S85A mutant enzymes was reduced by 44, 8, and 60%, respectively, when compared with the control. In vivo labeling experiments showed that the extent of phosphorylation of the S30A, S85A, and S30A,S85A mutant enzymes was reduced by 70, 17, and 83%, respectively. Phosphorylation of the S30A, S85A, and S30A,S85A mutant enzymes by protein kinase A in vitro was reduced by 60, 7, and 96%, respectively, and peptide mapping analysis of the mutant enzymes confirmed the phosphorylation sites in the enzyme. The incorporation of (3)H-labeled choline into phosphocholine and phosphatidylcholine in cells bearing the S30A, S85A, and S30A,S85A mutant enzymes was reduced by 56, 27, and 81%, respectively, and by 58, 33, and 84%, respectively, when compared with control cells. These data supported the conclusion that phosphorylation of choline kinase on Ser(30) and Ser(85) by protein kinase A regulates PC synthesis by the CDP-choline pathway.