Citations

ABSTRACT A 300-bp plasmid fragment of the hemagglutinin gene was used as target DNA to develop a rapid real-time LightCycler (Roche Applied Science, Indianapolis, Ind.) PCR assay for laboratory detection of smallpox virus. PCR primers and probes were designed specifically for detection of smallpox virus DNA, but all viruses of the genus Orthopoxvirus tested could be detected by use of the hemagglutinin gene target sequence. Base pair mismatches in the 204-bp amplicon allowed discrimination of cowpox virus (melting temperature [ T m ], 56.40°C), monkeypox virus ( T m , 56.24°C), and vaccinia virus ( T m , 56.72°C), including the Dryvax vaccine strain, from smallpox virus ( T m , 62.45°C) by melting curve analysis. The analytical sensitivity was 5 to 10 copies of target DNA per sample. The assay was specific for members of the genus Orthopoxvirus ; the DNAs of herpes simplex virus and varicella-zoster virus were not detected by the smallpox virus LightCycler PCR.

ABSTRACT The performance of the Amplified Mycobacterium Tuberculosis Direct (AMTD) test (Gen-Probe Inc., San Diego, Calif.) was assessed in a large tertiary care mycobacteriology laboratory. Both acid-fast smear-positive and smear-negative respiratory and nonrespiratory clinical specimens were analyzed. From February 1998 to 4 October 2001, AMTD assays were performed on 391 respiratory specimens and 164 nonrespiratory specimens. The AMTD assay was compared to the “gold standard” of combined culture and clinical diagnosis. The overall sensitivity for all specimens, including those for which no smear result was available, was 91.2%. The overall sensitivities of the assay, including acid-fast smear-positive and -negative specimens, were 97.8 and 77.3% for respiratory and nonrespiratory specimens, respectively. The corresponding specificities for respiratory and nonrespiratory specimens were 99.1 and 98.5%, respectively. The overall specificity for all specimens was 98.9%. Positive and negative predictive values were 93.9 and 99.7% and 91.7 and 96.4% for respiratory and nonrespiratory specimens, respectively. The time saved by using the AMTD test for making a diagnosis of tuberculosis instead of using culture was 8.99 days. Inhibitors to the AMTD assay were found in 3.1% of respiratory specimens and 3.1% of nonrespiratory specimens. The assay, used in a general mycobacteriology laboratory setting, represents an important advance in improving the speed and accuracy of diagnosis in the management of patients with tuberculosis.

ABSTRACT In vitro surveys of antimicrobial resistance among clinically important anaerobes are an important source of information that can be used for clinical decisions in the choice of empiric antimicrobial therapy. This study surveyed the susceptibilities of 556 clinical anaerobic isolates from four large medical centers using a broth microdilution method. Piperacillin-tazobactam was the only antimicrobial agent to which all the isolates were susceptible. Similarly, imipenem, meropenem, and metronidazole were highly active (resistance, <0.5%), whereas the lowest susceptibility rates were noted for penicillin G, ciprofloxacin, and clindamycin. For most antibiotics, blood isolates were less susceptible than isolates from intra-abdominal, obstetric-gynecologic, and other sources. All isolates of the Bacteroides fragilis group were susceptible to piperacillin-tazobactam and metronidazole, while resistance to imipenem and meropenem was low (<2%). For these same isolates, resistance rates (intermediate and resistant MICs) to ampicillin-sulbactam, cefoxitin, trovafloxacin, and clindamycin were 11, 8, 7, and 29%, respectively. Among the individual species of the B. fragilis group, the highest resistance rates were noted among the following organism-drug combinations: for clindamycin, Bacteroides distasonis and Bacteroides ovatus ; for cefoxitin, Bacteroides thetaiotaomicron, B. distasonis , and Bacteroides uniformis; for ampicillin-sulbactam, B. distasonis, B. ovatus , and B. uniformis ; and for trovafloxacin, Bacteroides vulgatus . For the carbapenens, imipenem resistance was noted among B. fragilis and meropenem resistance was seen among B. fragilis, B. vulgatus , and B. uniformis . With few exceptions all antimicrobial agents were highly active against isolates of Prevotella, Fusobacterium, Porphyromonas , and Peptostreptococcus . These data further establish and confirm that clinically important anaerobes can vary widely in their antimicrobial susceptibilities. Fortunately most antimicrobial agents were active against the test isolates. However, concern is warranted for what appears to be a significant increases in resistance to ampicillin-sulbactam and clindamycin.

ABSTRACT We compared the activities of linezolid (25 mg/kg of body weight, administered intraperitoneally every 8 h) and of vancomycin (25 mg/kg of body weight, administered intraperitoneally every 8 h) in a rat model of vanA vancomycin-resistant Enterococcus faecium experimental endocarditis. Results were expressed as median log 10 CFU per gram of vegetation after 3 days of treatment. The median log 10 CFU per gram of vegetation was 10.1 among 7 untreated control animals, 10.2 among 9 vancomycin-treated animals, and 7.9 among 10 linezolid-treated animals. Linezolid treatment was more active ( P < 0.05) than vancomycin treatment or no treatment.

ABSTRACT The activity of trovafloxacin was compared with those of vancomycin and penicillin in a model of Streptococcus sanguis species group (trovafloxacin MIC, 0.125 μg/ml) and Streptococcus mitis species group (trovafloxacin MIC, 0.125 μg/ml) experimental endocarditis. Rabbits with catheter-induced aortic valve vegetations were given no treatment, trovafloxacin at 15 mg/kg of body weight three times a day (t.i.d.), vancomycin at 15 mg/kg twice a day, or penicillin at 1.2 × 10 6 IU t.i.d. After 3 days of treatment, the animals were sacrificed; cardiac valve vegetations were aseptically removed and cultured quantitatively. Penicillin was as active as vancomycin as measured by in vivo clearance of bacteria. Trovafloxacin was less active ( P < 0.05) than vancomycin or penicillin against S. sanguis species group infection but had similar efficacy against S. mitis species group infection. Quinolones, despite MICs in the susceptible range, may not be active for serious infections caused by some viridans group streptococci.

ABSTRACT We have previously identified, in Paenibacillus popilliae , a 708-bp sequence which has homology to the sequence of the enterococcal vanA gene. We have performed further studies revealing five genes encoding homologues of VanY, VanZ, VanH, VanA, and VanX in P. popilliae . The predicted amino acid sequences are similar to those in VanA vancomycin-resistant enterococci: 61% identity for VanY, 21% for VanZ, 74% for VanH, 77% for VanA, and 79% for VanX. The genes in P. popilliae may have been a precursor to or have had ancestral genes in common with vancomycin resistance genes in enterococci. The use of P. popilliae biopesticidal preparations in agricultural practice may have an impact on bacterial resistance in human pathogens.

ABSTRACTWe have analyzed three cases of hepatitis C virus (HCV)-infected recipients who received blood from HCV-infected donors. Two recipients were exposed to two different HCV RNA-positive donors, and one was exposed to a single donor. All parental genomes from the actual infecting units of blood and the recipients were defined, and their presence in the follow-up serum samples was determined using sensitive strain-specific assays. The strain from one of the donors was found to predominate in all recipients' serum samples collected throughout the follow-up period of 10 to 30 months. In two recipients exposed to two infected donors, the strain from the second donor was occasionally found at very low level. However, the original recipients' strains were not detected. Our observations show that HCV-infected individuals can be superinfected with different strains, and this event may lead to eradication or suppression of the original infecting strain. Furthermore, our findings demonstrate that simultaneous exposure to multiple HCV strains may result in concomitant infection by more than one strain, although a single strain could rapidly establish its dominance. The results of the present study suggest the existence of competition among infecting HCV strains which determines the ultimate outcome of multiple HCV exposure.

ABSTRACTElderly individuals are at high risk for morbidity and mortality when infected with influenza virus. Vaccinations with inactivated virus are less effective in the elderly due to the declining competency of the aging immune system. We have explored whether immunological parameters predict poor anti-influenza virus vaccine responses and can be used as biological markers of immunosenescence. One hundred fifty-three residents of community-based retirement facilities aged 65 to 98 years received a trivalent influenza vaccine. Vaccine-induced antibody responses were determined by comparing hemagglutination inhibition titers before and 28 days after immunization. The composition of the T-cell compartment was analyzed by flow cytometry and the sizes of three T-cell subsets, CD4+CD45RO+cells, CD4+CD28nullcells, and CD8+CD28nullcells, were determined. Only 17% of the vaccine recipients were able to generate an increase in titers of antibody to all three vaccine components, and 46% of the immunized individuals failed to respond to any of the three hemagglutinins. The likelihood of successful vaccination declined with age and was independently correlated with the expansion of a particular T-cell subset, CD8+CD28nullT cells. The sizes of the CD4+CD45RO+memory T-cell and CD4+CD28nullT-cell subsets had no effect on the ability to mount anti-influenza virus antibody responses. Frequencies of CD8+CD28nullT cells are useful biological markers of compromised immunocompetence, identifying individuals at risk for insufficient antibody responses.

ABSTRACTThe role of NF-κB in the reactivation of human immunodeficiency virus (HIV) from latency in CD4 T lymphocytes is well documented. However, its role in driving HIV transcription in human macrophages, which contain a constitutive nuclear pool of NF-κB, is less well understood. In this study we have investigated the role that the constitutive pool of NF-κB and the NF-κBcis-acting motifs of the HIV long terminal repeat (LTR) play in regulating HIV transcription in human monocytic cells and primary macrophages. Inhibition of the constitutive nuclear pool of NF-κB (RelA and RelB) in the promonocytic U937 cell line using dominant-negative IκBα significantly decreases HIV replication. Moreover, it is demonstrated that in the differentiated monocytic cell line THP1, which contains a constitutive nuclear pool of NF-κB (RelB),an HIV provirus containing mutations of the κBcis-acting sites in the LTR is transcriptionally impaired. Reduction of the constitutive pool of NF-κB in human macrophages by an adenovirus vector expressing a dominant-negative IκBα also reduces HIV transcription. Lastly, mutation of the NF-κBcis-acting sites in the LTR of an R5 HIV provirus completely abrogates the first cycle of HIV transcription. These studies indicate that thecis-acting NF-κB motifs of the HIV LTR are critical in initiating HIV transcription in human macrophages and suggest that the constitutive nuclear pool of NF-κB is important in regulating HIV transcription in these cells.