Cytomegalovirus (CMV) infection and disease are important causes of morbidity and mortality in transplant recipients. For the purpose of developing consistent reporting of CMV outcomes in clinical trials, definitions of CMV infection and disease were developed and most recently published in 2002. Since then, there have been major developments in its diagnosis and management. Therefore, the CMV Drug Development Forum consisting of scientists, clinicians, regulators, and industry representatives has produced an updated version incorporating recent knowledge with the aim to support clinical research and drug development. The main changes compared to previous definitions are the introduction of a "probable disease" category and to incorporate quantitative nucleic acid testing in some end-organ disease categories. As the field evolves, the need for updates of these definitions is clear, and collaborative efforts between scientists, regulators, and industry can provide a platform for this work.

The gene glvA (formerly glv-1) from Bacillus subtilis has been cloned and expressed in Escherichia coli. The purified protein GlvA (449 residues, M r ‫؍‬ 50,513) is a unique 6-phosphoryl-O-␣-D-glucopyranosyl:phosphoglucohydrolase (6-phospho-␣-glucosidase) that requires both NAD(H) and divalent metal (Mn 2؉ , Fe 2؉ , Co 2؉ , or Ni 2؉) for activity. 6-Phospho-␣-glucosidase (EC 3.2.1.122) from B. subtilis cross-reacts with polyclonal antibody to maltose 6-phosphate hydrolase from Fusobacterium mortiferum, and the two proteins exhibit amino acid sequence identity of 73%. Estimates for the M r of GlvA determined by SDS-polyacrylamide gel electrophoresis (51,000) and electrospray-mass spectroscopy (50,510) were in excellent agreement with the molecular weight of 50,513 deduced from the amino acid sequence. The sequence of the first 37 residues from the N terminus determined by automated analysis agreed precisely with that predicted by translation of glvA. The chromogenic and fluorogenic substrates, p-nitrophenyl-␣-D-glucopyranoside 6-phosphate and 4-methylumbelliferyl-␣-D-glucopyranoside 6-phosphate were used for the discontinuous assay and in situ detection of enzyme activity, respectively. Site-directed mutagenesis shows that three acidic residues, Asp 41 may function as the catalytic acid (proton donor) and nucleophile (base), respectively, during hydrolysis of 6-phospho-␣-glucoside substrates including maltose 6-phosphate and trehalose 6-phosphate. In metal-free buffer, GlvA exists as an inactive dimer, but in the presence of Mn 2؉ ion, these species associate to form the NAD(H)-dependent catalytically active tetramer. By comparative sequence alignment with its homologs, the novel 6-phospho-␣-glucosidase from B. subtilis can be assigned to the nine-member family 4 of the glycosylhydrolase superfamily.The serendipitous discovery in 1964 (1, 2) of the bacterial phosphoenol pyruvate-dependent sugar phosphotransferase system (PEP-PTS) 1 by Roseman and colleagues represents a landmark in our understanding of carbohydrate transport by microorganisms (3, 4). Since the initial description in Escherichia coli, this phosphoryl group-transfer system (5, 6) has been established as the primary mechanism for the accumulation of sugars by bacteria from both Gram-negative (7, 8) and Gram-positive genera (9 -12). Operationally, the multi-component PEP-PTS (13) comprises both membrane-localized and cytoplasmic proteins that in concert catalyze the simultaneous phosphorylation and vectorial translocation of sugar across the cytoplasmic membrane. Catalytically, each PEP-PTS requires two general components (Enzyme I and HPr) that, allied with sugar-specific proteins (IIA, -B, and -C; for discussion, see Ref. 14), promote the sequential transfer of the high energy, phosphoryl moiety from PEP to the incoming sugar. Prior to catabolism via energy-yielding pathways, the intracellular disaccharide phosphates must first be hydrolyzed to their constituent hexose 6-phosphate and aglycone moieties. Several phosphoglycosylhydrolases (whose ge...

Traditionally, Streptococcus pneumoniae is identified in the laboratory by demonstrating susceptibility to optochin. Between 1992 and 1998, 4 pneumococcal isolates exhibiting optochin resistance were recovered from patients at Children's National Medical Center. Three of the 4 isolates consisted of mixed populations of optochin-resistant and -susceptible organisms. Both subpopulations had identical antibiograms, serotypes, and restriction fragment profiles. The other isolate was uniformly resistant to optochin. Resistant strains had MICs of optochin 4-30-fold higher than susceptible strains, belonged to different serotypes, and had dissimilar restriction fragment profiles, indicating clonal unrelatedness. Resistance arose from single point mutations in either the a-subunit (W206S) or the c-subunit (G20S, M23I, and A49T) of H(+)-ATPase. There is speculation of a possible association between exposure to antimalarial drugs and evolution of optochin resistance. alpha-Hemolytic streptococci resistant to optochin, particularly invasive isolates, should be tested for bile solubility or with an S. pneumoniae DNA probe before identification as viridans streptococci.

Enterococcus faecalis was tested for the ability to persist in mouse peritoneal macrophages in two separate studies. In the first study, the intracellular survival of serum-passaged E. faecalis 418 and two isogenic mutants [cytolytic strain FA2-2(pAM714) and non-cytolytic strain FA2-2(pAM771)] was compared with that of Escherichia coli DH5α by infecting BALB/c mice intraperitoneally and then monitoring the survival of the bacteria within lavaged peritoneal macrophages over a 72-h period. All E. faecalis isolates were serum passaged to enhance the production of cytolysin. E. faecalis 418, FA2-2(pAM714), and FA2-2(pAM771) survived at a significantly higher level (P = 0.0001) than did E. coli DH5α at 24, 48, and 72 h. Internalized E. faecalis 418, FA2-2(pAM714), and FA2-2(pAM771) decreased 10-, 55-, and 31-fold, respectively, over the 72-h infection period, while internalizedE. coli DH5α decreased 20,542-fold. The difference in the rate of survival of E. faecalis strains and E. coli DH5α was most prominent between 6 and 48 h postinfection (P = 0.0001); however, no significant difference in killing was observed between 48 and 72 h postinfection. In the second study, additional E. faecalisstrains from clinical sources, including DS16C2, MGH-2, OG1X, and the cytolytic strain FA2-2(pAM714), were compared with the nonpathogenic gram-positive bacterium, Lactococcus lactis K1, for the ability to survive in mouse peritoneal macrophages. In these experiments, the E. faecalis strains and L. lactis K1 were grown in brain heart infusion (BHI) broth to ensure that there were equal quantities of injected bacteria. E. faecalis FA2-2(pAM714), DS16C2, MGH-2, and OG1X survived significantly better (P < 0.0001) than did L. lactis K1 at each time point. L. lactis K1 was rapidly destroyed by the macrophages, and by 24 h postinfection, viable L. lactis could not be recovered. E. faecalis FA2-2(pAM714), DS16C2, MGH-2, and OG1X declined at an equivalent rate over the 72-h infection period, and there was no significant difference in survival or rate of decline among the strains. E. faecalis FA2-2(pAM714), MGH-2, DS16C2, and OG1X exhibited an overall decrease of 25-, 55-, 186-, and 129-fold respectively, between 6 and 72 h postinfection. The overall reduction by 1.3 to 2.27 log units is slightly higher than that seen for serum-passaged E. faecalis strains and may be attributable to the higher level of uptake of serum-passaged E. faecalis than of E. faecalis grown in BHI broth. Electron microscopy of infected macrophages revealed that E. faecalis 418 was present within an intact phagocytic vacuole at 6 h postinfection but that by 24 h the infected macrophages were disorganized, the vacuolar membrane was degraded, and the bacterial cells had entered the cytoplasm. Macrophage destruction occurred by 48 h, and the bacteria were released. In conclusion, the results of these experiments indicate that E. faecaliscan persist for an extended period in mouse peritoneal macrophages.

Symptomatic cytomegalovirus (CMV) disease has been the standard endpoint for clinical trials in organ transplant recipients. Viral load may be a more relevant endpoint due to low frequency of disease. We performed a meta-analysis and systematic review of the literature. We found several lines of evidence to support the validity of viral load as an appropriate surrogate end-point, including the following: (1) viral loads in CMV disease are significantly greater than in asymptomatic viremia (odds ratio, 9.3 95% confidence interval, 4.6-19.3); (2) kinetics of viral replication are strongly associated with progression to disease; (3) pooled incidence of CMV viremia and disease is significantly lower during prophylaxis compared with the full patient follow-up period (viremia incidence: 3.2% vs 34.3%; P < .001) (disease incidence: 1.1% vs 13.0%; P < .001); (4) treatment of viremia prevented disease; and (5) viral load decline correlated with symptom resolution. Based on the analysis, we conclude that CMV load is an appropriate surrogate endpoint for CMV trials in organ transplant recipients.

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Multidrug-resistant Streptococcus pneumoniae strains have emerged over the past decade at an alarming rate. The molecular mechanism of trimethoprim resistance was investigated in 5 pneumococcal strains isolated in the Washington, DC, area from patients with invasive infections. Cloning and sequencing of the trimethoprim resistance determinant from these pneumococci indicated that an altered chromosome-encoded dihydrofolate reductase (DHFR) was responsible for the observed resistance. Comparison of DHFR sequences from pneumococcal strains with various susceptibilities to trimethoprim, together with site-directed mutagenesis, revealed that substitution of isoleucine-100 with a leucine residue resulted in trimethoprim resistance. Hydrogen bonding between the carbonyl oxygen of isoleucine-100 and the 4-amino group of trimethoprim is proposed to play a critical role in the inhibition of DHFR by trimethoprim. This enzyme-substrate model should facilitate the design of new antibacterial agents with improved activity against S. pneumoniae.

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