Characterization of a novel human calicivirus that may be a naturally occurring recombinant

Summary.  We identified a Norwalk-like calicivirus (CV) whose genome likely was derived from naturally occurring recombination. This strain (Arg320) was detected by the EIA developed against recombinant Mexico virus (rMxV) capsids, but the viral RNA polymerase sequence was closer to Lordsdale virus, in a separate genetic cluster of Norwalk-like viruses. A 3.3 kb cDNA from the RNA polymerase region to the 3′ end of the genome of Arg320 was cloned and sequenced. The sequence demonstrated that the capsid region of Arg320 shared 95% amino acid identity with MxV, but 68% identity with Lordsdale virus, while the RNA polymerase region shared 95% identity with Lordsdale virus, but 87% identity with MxV. Pair-wise sequence comparisons identified a potential recombination site at the polymerase/capsid junction. This is the first example of a naturally occurring recombinant in the CV family. Further studies to search for and characterize other strains may be necessary for understanding the genetic diversity of the family. Received April 26, 1999/Accepted July 15, 1999     

DNA fingerprinting of Enterococcus faecium by pulsed-field gel electrophoresis may be a useful epidemiologic tool.

Pulsed-field gel electrophoresis was used to compare 34 isolates of Enterococcus faecium from six different geographic locations. This procedure generated an average of 13 discernible fragment bands per isolate (range, 10 to 19 fragment bands) of 34 to 485 kb. The resulting restriction endonuclease digestion patterns were quite heterogeneous and were able to differentiate 27 of 34 isolates from each other, as defined by one or more mismatched fragment bands. Five patterns were shared by two or more isolates, and each set of isolates with matching patterns (shared pattern) originated in the same medical center, suggesting a common epidemiologic background, including highly penicillin resistant isolates in Richmond and Philadelphia. We conclude that pulsed-field gel electrophoresis of DNA digested with low-frequency-cleavage restriction enzymes offers a relatively simple method of comparing E. faecium for the purpose of epidemiologic study.     

Determination of amphotericin B, liposomal amphotericin B, and amphotericin B colloidal dispersion in plasma by high-performance liquid chromatography.

Amphotericin B is a potent polyene antifungal drug for intravenous treatment of severe infections. It is used as amphotericin B-deoxycholate and in order to reduce amphotericin B toxicity as lipid-formulated complex (liposomal or colloidal dispersion). A sensitive and specific analytical method is presented for the separation of lipid-complexed and plasma protein-bound amphotericin B in human heparinized plasma. This separation, which is required for pharmacokinetic studies, is achieved by solid-phase extraction (SPE) via Bond Elut C18. The protein-bound amphotericin B has a higher affinity to the SPE material and is therefore retained, whereas the lipid-complexed amphotericin B is eluted in the first step. The recovery of the SPE was >75% for high concentrations and >95% for low concentrations. Quantification was performed by reversed-phase HPLC using a LiChrosorb-RP-8 column, UV detection (lambda=405 nm) and a mixture of acetonitrile-methanol-0.010 M NaH2PO4 buffer (41:10:49, v/v) as mobile phase. The retention time for amphotericin B under the given conditions was 6.7 min. The calibration curves were found to be linear (r > or = 0.999) in two different ranges (5.0-0.50 microg/ml and 0.50-0.005 microg/ml). Intra- and inter-day precision and accuracy fulfilled the international requirements. No interference from other drugs (typical broad medication for intensive-care patients) or common plasma components was detected in >400 samples analyzed.     

Measurement of the local aortic stiffness by a non-invasive bioelectrical impedance technique

Aortic stiffness measurement is well recognized as an independent predictor of cardiovascular mortality and morbidity. Recently, a simple method has been proposed for the evaluation of the local aortic stiffness (AoStiff) using a non-invasive bioelectrical impedance (BI) technique. This approach relies on a novel interpretation of the arterial stiffness where AoStiff is computed from the measurement of two new BI variables: (1) the local aortic flow resistance (AoRes) exerted by the drag forces onto the flow; (2) the local aortic wall distensibility (AoDist). Herein, we propose to detail and compare these three indices with the reference pulse wave velocity (PWV) measurement and the direct assessment of the aortic drag forces (DF) and distensibility (DS) obtained by the magnetic resonance imaging technique. Our results show a significant correlation between AoStiff and PWV (r = 0.79; P < 0.0001; 120 patients at rest; mean age 44 ± 16 years), and also between AoRes and DF (r = 0.95; P = 0.0011) and between AoDist and DS (r = 0.93; P = 0.0022) on eight patients at rest (mean age 52 ± 19 years). These first results suggest that local aortic stiffness can be explored reliably by the BI technique.     

Measurement of bacterial concentration fractions in polymicrobial mixtures by Raman microspectroscopy

Relative concentrations of Streptococcus mutans and Streptococcus sanguis are important parameters in the study of dental caries, but current methods of measuring these concentrations are time consuming and prone to inaccuracies. We investigate the use of Raman spectroscopy for measuring relative concentrations of these two bacterial species in solid mixtures. To our knowledge, this is the first time Raman spectroscopy has been used to analyze bacterial mixtures rather than to identify the species of a pure colony. Mixtures of the two streptococcal species in various ratios are measured for 200 s using a home-built Raman microscope. Spectral correlations with bacterial content were identified via partial least-squares analysis. The relative concentrations of S. mutans in subsequent samples are predicted with a root mean squared error below 5%. In clinical plaque samples, this sort of accuracy would enable discrimination between normal and dangerously elevated levels of S. mutans. Samples with and without salivary proteins are predicted with equal accuracy. This result shows the potential of Raman spectroscopy for analyzing mixed populations of bacteria, such as those that occur in oral plaques.     

Restriction endonuclease analysis of Staphylococcus epidermidis DNA may be a useful epidemiological marker.

We compared the epidemiological markers of 13 Staphylococcus epidermidis strains isolated from an adult inpatient during a febrile episode and 23 S. epidermidis strains isolated during a presumptive outbreak of nosocomial infection in a neonatal ward. The total DNA restriction endonuclease analysis (REA) was processed along with the following conventional markers: biotyping, serotyping, phage typing, antibiotic susceptibility profiles, and plasmid profiles. The REA method was reproducible, giving stable results both in vitro and in vivo. For the hospitalized adult patient, the conventional markers of the 13 strains were concordant and the restriction profiles were identical. Five restriction groups were demonstrated during the course of the outbreak. Within two of the groups, the identities of all of the markers were used to verify whether all of the isolates belonged to the same cell clone. In a third group, combined analysis of the conventional markers and REA had to be used to demonstrate isolate similarity. On the other hand, in another group, none of the markers were similar; interpretation was not easy. An epidemiological study of S. epidermidis infections in hospitals must take into account all of the epidemiological markers: biotypes, serotypes, phage types, antibiograms, plasmid profiles, and REA.     

Is there a predisposition for meiotic non-disjunciton that may be detected by mitoitc hyperploidy?

The aneuploidy frequencies have been studied in vitro in lymphocytes from couples with recurrent abortions, from parents of a trisomic child and from control couples with at least two normal children; c-heterochromatin variants have been analysed on the same samples by length measurements. A significant increase of hyperploid cells has been observed in the lymphocytes from parents of a trisomic child and couples with recurrent abortions as well. However, no consistent correlation has been found so far between c-heterochromatin variants and an increase of aneuploidy.     

Anatomic variations of popliteal artery that may be a reason for entrapment

Purpose  

The aim of this study was to demonstrate some anatomic variations of popliteal artery and its surrounding structures that may be important especially for popliteal artery entrapment (PAE) syndrome.     

Comparison of the minimum fungicidal concentration of amphotericin B determined in filamentous fungi by macrodilution and microdilution methods.

Minimum fungicidal concentration (MFC) determination could be useful in severe fungal infections in immunocompromised patients. No reference tests to determine the MFC are available, and both macro- and microdilution methods are commonly used. In this study, discrepancies between minimum inhibitory concentrations (MICs) and MFCs of amphotericin B against 58 isolates of filamentous fungi other than Aspergillus (46 Fusarium spp., six Paecilomyces spp. and six Scopulariopsis spp.), obtained with macro- and microdilution methods, were evaluated. Additionally, the agreement between MFCs obtained by both methods were analyzed. In general MFCs were higher than the corresponding MICs overall using the macrodilution method. MFCs were more than one dilution higher than MICs in 52.3% of the cases in the macrodilution test and in 20.5% of the cases in microdilution test. The degree of agreement between MFCs obtained with the two methods was of 70.4% (Kappa coefficient of 0.5). In general, the macrodilution method showed higher MFC values than the microdilution method. Differences of up to six drug dilutions were observed between MFCs obtained by both methods.     

Calcitonin may be a useful therapeutic agent for osteoclastogenesis syndromes involving premature eruption of the tooth

Tooth eruption is a complex and tightly regulated process that involves cells of the tooth organ and the surrounding alveolus. Recent researches have shown that tooth eruption depends on the presence of osteoclasts to create an eruption pathway through the alveolar bone. The most important physiologic role likely being at the eruptive site, in the formation of osteoclasts through signaling via the RANKL/OPG pathway. Calcitonin is an endogenous inhibitor of osteoclast development and function and thus of bone resorption. Specific calcitonin receptors are expressed on osteoclasts and their activation leads to the inhibition of osteoclast development and functions. Recent concepts about inhibiting osteoclastogenesis of calcitonin is that RANKL-induced osteoclastogenesis were blocked by the endogenous decoy receptor osteoprotegerin and were also strongly reduced by calcitonin, we hypothesize that calcitonin may has anti-eruption properties. For the clinical point of view, we can inject calcitonin in the oral mucosa of the affected tooth to inhibit bone resorption, then to facilitate root forming which may useful to premature eruption of tooth and short root anomaly disease (SRA) caused by every reasons such as hypoplasia of teeth root (HTR), Singleton-Mertern syndrome (SMS), infection and iatrogenic factors, etc.     

PCR monitoring of response to liposomal amphotericin B treatment of systemic candidiasis in neutropenic mice.

When a diagnosis of invasive candidiasis has been made, treatment with toxic fungicidal agents is inevitable. The crucial decision of when to stop such treatment is difficult to make, because cultures are often negative despite ongoing invasive candidiasis and can therefore not be used as a reliable parameter of effective therapy. In the present study, the use of PCR in monitoring the therapeutic efficacy of antifungal treatment with liposomal amphotericin B was evaluated by using neutropenic mice with systemic candidiasis. Blood cultures of infected mice treated with different doses of liposomal amphotericin B were only positive at the early onset of the infection process and became sterile within 3 days; this was true even with mice treated with 1 mg of liposomal amphotericin B per kg of body weight that experienced a relapse of infection 14 days later. A significant correlation between presence of Candida albicans in the kidneys and PCR results obtained with blood was demonstrated. Thus, PCR results obtained with blood samples correlated well with the therapeutic efficacy of antifungal treatment.     

Inhibition of human neutrophil chemotaxis and chemiluminescence by amphotericin B.

The effect of the antifungal drugs amphotericin B, fluocytosine, miconazole, griseofulvin, and nystatin on the chemotactic responsiveness of human neutrophils was studied. Amphotericin B in a concentration of 2 mug/ml inhibited chemotatic responsiveness, and in a concentration of 5 mug/ml it also inhibited chemiluminescence. The inhibition of chemotaxis could be reversed by washing the cells. The other antifungal drugs did not inhibit chemotaxis even in concentrations much higher than those obtained in human serum during treatment.     

Suppression of immunological responses in mice by treatment with amphotericin B.

The polyene antibiotic amphotericin B (AmB) caused a marked suppression of the cell-mediated immune response in mice. Similar treatment did not effect the humoral antibody response. The immunosuppressive property of the drug was related to its ability to inhibit the manifestation rather than the induction phase of the delayed-type hypersensitivity response. In vitro AmB suppressed mitogen-induced lymphocyte proliferation. The drug seemed to act at the proliferative phase of the response. Results presented show that the T cell response was much more sensitive to the action of AmB than was the B cell response. During AmB chemotherapy consideration must be given to the immunosuppressive properties of this drug.     

Evidence that amphotericin B mediates reactivation of latent Epstein-Barr virus in Hodgkin's lymphoma allowing cytotoxicity by acyclovir

This brief communication focuses on aspects of a recent case report (Yonsei Med J 2005;46:425-30) on a full and sustained remission of Hodgkin's lymphoma (HL) after a single day of chemotherapy. A septic episode required stopping chemotherapy and starting amphotericin B and acyclovir. Remission evidence was seen within days of starting these. A review of research supporting the notion that amphotericin B can reactivate latent Epstein-Barr virus and thus allow acyclovir to kill infected HL cells is given. Experimental work is required to confirm or refute this possibility. If successful, amphotericin B and acyclovir treatment could be extended to other EBV-driven cancers such as Burkitt's lymphoma, nasopharyngeal carcinoma and the occasional EBV-related epithelial cancer of the breast, colon, prostate, and others.     

CD16+ monocytes in breast cancer patients: expanded by monocyte chemoattractant protein-1 and may be useful for early diagnosis

Human peripheral blood monocytes are a heterogeneous population, including CD14⁺CD16^‐‘classical’ monocytes and CD14⁺CD16⁺‘proinflammatory’ monocytes. CD16⁺ monocytes are expanded in various inflammatory conditions. However, little is known about the CD14⁺CD16⁺ monocytes in patients with breast cancer. We detected CD14⁺CD16⁺ monocytes in 96 patients with breast cancer and 54 control subjects using flow cytometry. Receiver‐operating characteristic (ROC) curve analysis was used to determine the feasibility of CD14⁺CD16⁺ monocytes as an indicator for diagnosis of breast cancer. We found that the frequency of CD14⁺CD16⁺ monocytes showed a significantly greater increase in breast cancer patients than in controls (16·96% versus 10·84%, P < 0·0001). The area under the ROC curve for CD14⁺CD16⁺ monocytes was 0·805 [95% confidence interval (95% CI): 0·714–0·877, P = 0·0001]. Furthermore, the levels of CD16⁺ monocytes were significantly negatively associated with the tumour size and pathological staging. In vitro, we showed that CD14⁺CD16⁺ monocytes were expanded significantly when the purified CD14⁺ monocytes were exposed to Michigan Cancer Foundation (MCF)‐7 cells‐conditioned medium (MCF‐CM) or, separately, to monocyte chemotactic protein 1 (MCP‐1). Neutralizing antibodies against MCP‐1 inhibited the expansion of CD14⁺CD16⁺ monocytes by MCF‐CM. Collectively, our findings indicated that MCP‐1 can expand CD14⁺CD16⁺ monocytes in patients with breast cancer. Furthermore, the CD14⁺CD16⁺ monocyte may be a useful indicator in early diagnosis of breast cancer.     

Enhancement of infectivity of encephalomyocarditis virus RNA by amphotericin B methyl ester.

The methyl ester of amphotericin B (AmBME), a macrolide polyene antibiotic, enhanced the infectivity of encephalomyocarditis (EMC) virus RNA for L929 cells. AmBME alone (100 microgram/ml) resulted in increases in EMC virus RNA infectivity of 10- to 100-fold. Addition of DEAE dextran at concentrations (5 microgram/ml), which alone slightly suppressed EMC virus RNA infectivity, further augmented the effects of AmBME (augmentation in infectivity up to 750-fold). AmBME did not inhibit RNase, did not enhance EMC virus infectivity and increased infectivity of EMC virus RNA which was already cell-associated. The polyenes are probably acting by increasing intracellular penetration of polyribonucleotides.     

Survey of amphotericin B susceptibility of Candida clinical isolates determined by Etest.

BACKGROUND AND PURPOSE: The minimal inhibitory concentrations (MICs) of amphotericin B (AmB) determined by the National Committee for Clinical Laboratory Standards (NCCLS; NCCLS document M27-A) broth dilution method are in a relatively narrow ranges and this may lead to underestimation of the AmB-resistant rate in clinical isolates. We evaluated in vitro susceptibility of clinical isolates of Candida spp. to AmB using Etest and determined the distribution of AmB MICs in different species. METHODS: We used the Etest (AB Biodisk, Solna, Sweden) to evaluate the MICs of Candida isolates randomly collected during 2001-2003 in a teaching hospital. RESULTS: Of the 572 isolates evaluated, Candida albicans (50.7%) was the most common species, followed by Candida tropicalis (23.9%), Candida parapsilosis (13.1%), Candida glabrata (9.4%), Candida krusei (1.9%), and Candida guilliermondii (0.9%). The majority of isolates were from blood (85%). The minimal concentrations of AmB required to inhibit 50%/90% of the isolates (MIC(50)/MIC(90)) were 0.19/0.38 microg/mL for C. krusei, 0.125/0.38 microg/mL for C. glabrata, 0.094/0.25 microg/mL for C. tropicalis, 0.032/0.19 microg/mL for C. albicans, 0.016/0.125 microg/mL for C. parapsilosis, and 0.023/0.032 microg/mL for C. guilliermondii. Only 1 blood isolate of C. glabrata was resistant to AmB (MIC > or =1 microg/mL) [0.17%]. 18.2% of isolates were less susceptible to AmB (MIC > or =0.19 microg/mL) with the highest rates for C. krusei (63.6%), followed by C. glabrata (37.0%), C. tropicalis (29.9%), C. albicans (11.0%), C. parapsilosis (5.3%), and C. guilliermondii (0%). More isolates collected from patients with hematologic malignancy were less susceptible to AmB than those collected from those with other diseases (30.5% vs 15.4%, p<0.05). CONCLUSION: This study demonstrated that AmB resistance remains rare at this hospital in Candida clinical isolates despite increasing use of this agent during the past 4 decades.