Evaluation of an Immunochromatographic Assay for Rapid Detection of Penicillin-Binding Protein 2a in Human and Animal Staphylococcus intermedius Group, Staphylococcus lugdunensis, and Staphylococcus schleiferi Clinical Isolates.

The performance of a rapid penicillin-binding protein 2a (PBP2a) detection assay, the Alere PBP2a culture colony test, was evaluated for identification of PBP2a-mediated beta-lactam resistance in human and animal clinical isolates of Staphylococcus intermedius group, Staphylococcus lugdunensis, and Staphylococcus schleiferi. The assay was sensitive and specific, with all PBP2a-negative and PBP2a-positive strains testing negative and positive, respectively.

Draft Genome Sequence of the Pandoraea apista LMG 16407 Type Strain.

Pandoraea species, in particular Pandoraea apista, are opportunistic, multidrug-resistant pathogens in persons with cystic fibrosis (CF). To aid in understanding the role of P. apista in CF lung disease, we used Illumina MiSeq and nanopore MinION technology to sequence the whole genome of the P. apista LMG 16407(T).

Prevalence of blaZ gene types and the inoculum effect with cefazolin among bloodstream isolates of methicillin-susceptible Staphylococcus aureus.

We sought to define the prevalence of blaZ gene types and the inoculum effect to cefazolin among methicillin-susceptible Staphylococcus aureus (MSSA) bloodstream infections. The blaZ gene was present in 142/185 (77%) isolates. A total of 50 (27%) isolates had a ≥4-fold increase in the cefazolin MIC from a standard to a high inoculum, and 8 (4%) demonstrated a nonsusceptible cefazolin MIC, all type A blaZ strains. The efficacy of cefazolin in the presence of the inoculum effect requires further study.

Weissella confusa bacteremia in a liver transplant patient with hepatic artery thrombosis.

A 54-year-old man with a history of nonalcoholic steatohepatitis and hepatocellular carcinoma presented 2 months after an orthotopic liver transplant with fever and abdominal pain. Two weeks earlier, he had an hepatic artery thrombosis and a biliary stricture, for which an hepatic artery stent and a biliary stent were placed. Laboratory workup was significant for leukocyte count of 7800/mcL with 92% segmented neutrophils, hemoglobin 9.4 g/dL, alanine aminotransferase 98 U/L, aspartate aminotransferase 72 U/L, alkaline phosphatase 358 U/L, albumin 2.8 mg/dL, and total bilirubin 1.6 mg/dL. A computed tomography scan of the abdomen and pelvis revealed multiple small fluid collections in the liver consistent with bilomas, and an hepatic angiogram showed complete occlusion of the common hepatic artery. Two sets of blood cultures were positive for an organism initially identified by MicroScan(®) analysis as an α-hemolytic Streptococcus species that was resistant to vancomycin. Further testing confirmed the organism as Weissella confusa 2 days later. W. confusa is a gram-positive coccobacillus that may be misidentified as a Lactobacillus when cultured. It is commonly found in sewage, carrots, sugar cane, fermented foods, and intestinal flora. Although only 4 cases of clinical infection with W. confusa have been described previously, W. confusa has been isolated from the stool of liver transplant patients, and may be an underreported cause of infection owing to improper identification. As it can cause clinical infection in these immunosuppressed hosts, identification of this organism is paramount because it is vancomycin resistant, and incorrect identification could lead to improper antimicrobial selection and ultimately worsened patient morbidity or mortality.

The use of hepatitis C viral RNA levels in liver tissue to distinguish rejection from recurrent hepatitis C.

Persistence of hepatitis C virus (HCV) after orthotopic liver transplantation is almost universal in HCV-infected patients. Histological examination of liver biopsy specimens can be variable in distinguishing between recurrent hepatitis C and acute cellular rejection. The purpose of this study is to determine whether hepatic HCV RNA levels can be used to distinguish rejection from recurrent HCV by determining whether hepatic HCV RNA levels correlate with histological characteristics and clinical course. Seventy-two biopsy specimens were evaluated from 36 liver transplant recipients with HCV and elevated liver-related enzyme levels. Based on histological findings and clinical response to therapy, patients were defined as belonging to 1 of 5 groups: (1) definite rejection, (2) probable rejection, (3) indeterminate findings, (4) probable HCV, and (5) definite HCV. Hepatic HCV RNA was quantified using the Amplicor Monitor assay (Roche Diagnostic Systems Inc, Branchburg, NJ). There was a difference across groups in HCV RNA levels (P =.046). The median HCV RNA level was 10,695 copies/mg of tissue DNA in the definite-HCV group compared with 1,024 copies/mg of tissue DNA in the definite-rejection group. Using pairwise comparisons, significant differences were found between definite HCV and definite rejection, probable HCV and definite rejection, probable HCV and probable rejection, and probable HCV and indeterminate. Our findings support the following conclusions. (1) In liver transplant recipients, hepatic HCV RNA levels are statistically greater in patients with recurrent HCV than rejection, although there is considerable overlap between groups. (2) Patients with low HCV RNA levels were unlikely to have recurrent HCV. (3) Patients with minimal and indeterminate findings on biopsy (group 3) had low HCV RNA levels.

Tissue quantification of hepatitis C virus RNA with morphologic correlation in the diagnosis of recurrent hepatitis C virus in human liver transplants.

BACKGROUND: Histologic findings and liver enzymes in liver transplants are often non-diagnostic of recurrent hepatitis C virus (HCV) disease. In addition, the relationship between HCV replication and the presence of recurrent HCV hepatitis after liver transplantation remains unclear. We studied liver transplant recipients to determine if quantitation of HCV RNA in liver tissue by reverse transcriptase-polymerase chain reaction (RT-PCR) correlates with histopathologic disease and/or liver enzymes. METHODS: Twenty-six patients who received liver transplants for HCV infection were evaluated. Four sequential biopsies were analyzed for each patient. HCV RNA was extracted and quantified using the Amplicor HCV Monitor Test. Histologic examination and RNA quantitation were blinded. All available liver enzymes on the day of liver biopsy were analyzed. RESULTS: HCV RNA quantity in liver tissue was significantly increased at the time of clinically-suspected recurrence (P < .0001). HCV RNA levels were highest in biopsies with lobular hepatitis and nonspecific inflammation, followed by biopsies with cytomegalovirus infection, chronic hepatitis, and acute cellular rejection. HCV RNA quantity had a significant correlation with increasing portal inflammation (P = .0002), decreasing amount of interface hepatitis (P = .0333), and presence of acidophilic bodies (P = .0316). Increasing HCV RNA levels significantly correlated with decreasing number of episodes of treated rejection. HCV RNA quantity did not correlate with other histologic features or liver enzymes. CONCLUSIONS: HCV RNA levels are highest at the time of active hepatocellular destruction. Elevated HCV RNA indicates recurrence. HCV RNA quantitation may be a useful diagnostic test for determining recurrent disease and distinguishing it from other causes of inflammation, such as rejection.

Replication of human cytomegalovirus in human retinal glial cells.

PURPOSE: The purpose of these studies was to characterize the replication cycle of human cytomegalovirus (HCMV) in human retinal glial cells in vitro. METHODS: Cultured human retinal glial cells were exposed to HCMV strain AD169 or low-passage clinical isolates for a 2-hour adsorption period and then incubated in the appropriate growth medium at 37 degrees C. Cultures were examined by microscopy for cytopathic effect and by immunofluorescence staining using monoclonal antibodies directed against immediate-early, early, and late HCMV proteins. Viral DNA was analyzed by field inversion gel electrophoresis and detected using Southern blot analysis or the polymerase chain reaction. RESULTS: Immunocytochemical staining revealed that the glial cells expressed all three classes of HCMV proteins and that infectious virus could be transferred from the medium of the infected cultures to susceptible MRC-5 cell monolayers. Less than 1% of the glial cells expressed the S-phase enzyme, thymidine kinase, at the time of infection compared to MRC-5 fibroblasts, of which 81% expressed it. Progeny virus was found to be highly cell associated in glial cells (80%) at peak virus titer compared to MRC-5 cells (39% cell associated at peak titer). Four low-passage clinical isolates of HCMV from patients with acquired immune deficiency virus also productively infected cultures of human retinal glial cells. Field inversion gel electrophoresis of HCMV-infected glial cell lysates was performed to identify the replicative forms of DNA. Southern blots probed with HCMV-specific probes showed that HCMV DNA replication proceeds through high molecular weight intermediates before forming the 230-kb unit length genome. CONCLUSIONS: The full permissive replication of HCMV in human retinal glial cells indicates that glial cells are a likely site of HCMV replication in the retina and thus may play an important role in the pathogenesis of HCMV retinitis.

Maintenance of replicative intermediates in ganciclovir-treated human cytomegalovirus-infected retinal glia.

OBJECTIVES: To characterize the molecular structure of the human cytomegalovirus (HCMV) DNA maintained in cultures of human retinal glia following ganciclovir treatment and to determine the biological activity of the DNA. METHODS: Cultures of human retinal glia were established, infected with HCMV, treated with ganciclovir, and embedded in agarose, and the viral DNA was analyzed by field inversion gel electrophoresis. RESULTS: The HCMV DNA was found to persist in cultures of infected, ganciclovir-treated retinal glial cells in the form of replicative intermediates. After removal of ganciclovir, processed forms of DNA in the 500-to 1000-kilobase range were found as well as 230-kb unit length genome. Infectious virus was recovered after termination of ganciclovir treatment. CONCLUSION: The data are consistent with the concept that ganciclovir's virostatic nature permits maintenance of HCMV DNA in retinal glia in a biologically active form that is capable of replication after removal of the drug.

Human herpesvirus 6 (HHV-6)-associated dysfunction of blood monocytes.

HHV-6 is a recently described member of the herpesvirus family. HHV-6-associated marrow failure and interstitial pneumonitis where macrophages are the primary infected cell type have been described in marrow transplant patients (Carrigan, 1991; Drobyski et al., 1993). In recent studies we have shown that exposure of normal human marrow to HHV-6GS (a type A strain) or several type B strains resulted in suppression of growth factor induced outgrowth of macrophages by > 90% (Burd and Carrigan, 1993). Additional experiments using HHV-6GS to characterize the effects of the virus on peripheral blood monocytes showed that the respiratory burst capacity of these cells as determined by luminol-enhanced chemiluminescence using phorbol myristate acetate as a trigger was decreased by 83% +/- 13% in a series of 5 experiments. The decreased respiratory burst was evident as early as 15 min after exposure to virus. Experiments in which cells were separated on a fluorescence activated cell sorter prior to respiratory burst assay showed that the response was mediated solely by peripheral blood monocytes. The respiratory burst response of virus-exposed cells to opsonized zymosan was not affected, indicating that the virus may selectively interfere with the protein kinase C pathway of cellular activation. Ultracentrifugation of stock material to remove infectious virus showed that the suppressive factor was associated with the supernatant fraction. These findings suggest that HHV-6 infection may be associated with a defect in one of the major monocyte activation pathways, and this could be of importance with respect to persistent infection by HHV-6 in immune compromised patients.

Fibronectin and proteolytic fragments of fibronectin interfere with the adhesion of Staphylococcus epidermidis to plastic.

The adhesion of five strains of slime-positive Staphylococcus epidermidis to plastic microwells was significantly diminished (P < 0.005) in a concentration-dependent fashion when wells were previously coated with increasing concentrations (1.6-13.1 micrograms cm-2) of human fibronectin (FN). The adhesion of four of five strains was significantly reduced when wells were coated with 3.2 micrograms cm-2 of FN and at concentrations > or = 6.5 micrograms cm-2 the adhesion of all slime-positive strains was significantly reduced. The coating of microwells with chymotryptic fragments of FN containing the heparin-binding, gelatin-binding, or cell-binding domains also reduced bacterial adhesion but none of the fragments exceeded the anti-adhesive activity of intact FN. A comparison of FN-coated or albumin-coated microwells showed that both proteins caused a significant reduction in the adhesion of test strains to plastic but that the anti-adhesive activity of FN was greater than albumin at all concentrations tested. The adhesion of the slime-negative phase variant of one of the test strains to plastic was neither enhanced nor reduced by FN coating indicating that the production of an exopolysaccharide by Staph. epidermidis influences interactions with protein-coated surfaces. These results support the contention that FN does not mediate the adhesion of all strains of Staph. epidermidis to plastic surfaces.

Human herpesvirus-6-associated suppression of growth factor-induced macrophage maturation in human bone marrow cultures.

Cultures of marrow mononuclear cells were exposed to medium derived from cell cultures infected with several different strains of human herpesvirus-6 (HHV-6) immediately before the addition of either of two growth factors, granulocyte-macrophage colony-stimulating factor and interleukin-3. Exposure to any of the viral preparations suppressed the outgrowth of nonspecific esterase-positive adherent macrophages induced by the factors by more than 90%. The nonadherent cell populations in the infected cultures were numerically similar to those in uninfected control cultures, demonstrating the absence of a nonspecific cytotoxic effect of the viral materials. Infectious virus was not necessary for the macrophage outgrowth suppression. These findings suggest that HHV-6 either encodes or induces a soluble mediator or mediators that can interfere with the responses of bone marrow to growth factors and possibly block the normal differentiation of macrophages from marrow precursors.

Human herpesvirus-6 (HHV-6) infection in allogeneic bone marrow transplant recipients: evidence of a marrow-suppressive role for HHV-6 in vivo.

Sixteen adults were studied for the first 100 days after allogeneic bone marrow transplant to assess the pathogenic role of human herpesvirus-6 (HHV-6) infection in patients with unexplained febrile illnesses. HHV-6 was directly isolated from the blood of 6 patients. Analysis of the clinical courses of these 16 patients revealed otherwise unexplained posttransplant marrow suppression in 5 patients. Idiopathic marrow suppression occurred more frequently in patients with concurrent HHV-6 viremia (4/6) than in those from whom HHV-6 was not isolated from peripheral blood (1/10, P < .05). An etiologic role for the virus was also supported by isolation of HHV-6 from the bone marrow of all 4 patients at the time of marrow suppression and by in vitro colony-forming unit (cfu) assays that demonstrated that HHV-6 could inhibit cfu-granulocyte-macrophage and burst-forming unit-erythroid growth from human bone marrow. By restriction enzyme mapping, all clinical isolates were type B, suggesting that bone marrow transplant recipients may be preferentially infected with and reactivate this HHV-6 subtype. This study implicates HHV-6 as a novel cause of bone marrow suppression in marrow transplant recipients.

The effects of magnesium, calcium, EDTA, and pH on the in vitro adhesion of Staphylococcus epidermidis to plastic.

The effects of increasing concentrations of magnesium (Mg2+), calcium (Ca2+) or EDTA, and pH on the adhesion of five slime-positive strains of Staphylococcus epidermidis (Se+) to plastic were examined using an in vitro microwell assay. The addition of Mg2+ (as either MgSO4 or MgCl2) to the bacterial suspension in concentrations as low as 16 microM significantly enhanced the adhesion of all test strains to plastic (P < 0.001). Similarly, the addition of Ca2+ (as CaCl2) in concentrations exceeding 128 microM produced a significant increase in the adhesion of all test strains, but not to the extent observed with Mg2+. In contrast, the adhesion of all test strains to plastic was significantly reduced in the presence of EDTA at concentrations greater than 8 mM. However, EDTA in concentrations as low as 0.25 mM caused a significant decrease in the adhesion of two strains of Se+. The effect of pH was variable, but at a pH of 5.0 and 6.0, the adhesion of all test strains was significantly reduced compared to control values at a pH of 7.0. Two strains showed a significant increase in adhesion at a pH of 8.0. We also compared the effects of these variables on the adherence of a slime-negative phase variant derived from a slime-positive parent strain. With the exception of pH, the adhesion of both strains in response to increasing divalent cations or EDTA was similar. These data indicate that, in addition to hydrophobic interactions, ligand-specific binding, and slime production, pH and divalent cations, especially Mg2+, are important determinants of the adhesion of S. epidermidis to plastic surfaces in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of human intravenous immune globulin on diarrhea caused by Shiga-like toxin I and Shiga-like toxin II in infant rabbits.

Shiga toxin and the related Shiga-like toxins (SLT), produced by Escherichia coli, can cause hemorrhagic colitis and hemolytic uremic syndrome (HUS). Human intravenous immune globulin (HIVIg) blocks the cytotoxicity of some SLTs in vitro. To examine the ability of HIVIg to modify disease caused by Shiga-like toxin I or Shiga-like toxin II (SLT-I or SLT-II), we injected 3-day-old rabbits intraperitoneally with SLT-containing cell-free supernatants from Escherichia coli O157: H7. A subset of rabbits was treated with subcutaneous HIVIg. All rabbits given 10(4) CD50 of SLT-I developed severe diarrhea, and 5 died. When HIVIg 500 mg/kg was given in addition to SLT-I, only 6 of 18 rabbits (33.3%) developed diarrhea (P < 0.0001), and 1 died. HIVIg 500 mg/kg or 1,000 mg/kg protected against diarrhea when given one hour prior to toxin. HIVIg 1,000 mg/kg was protective when administered one hour after toxin, but not at 6 or 24 hr. Seventeen of 18 rabbits given 10(6) CD50 of SLT-II developed severe diarrhea, and 4 died. In contrast to SLT-I-associated disease, HIVIg had no effect on diarrhea in rabbits given SLT-II. We conclude that HIVIg protects infant rabbits from diarrhea and death caused by intraperitoneally administered SLT-I, but does not affect the course of SLT-II-associated illness.

In vitro measurement of the adherence of Staphylococcus epidermidis to plastic by using cellular urease as a marker.

A rapid and sensitive in vitro assay was developed to quantitatively assess the adherence of Staphylococcus epidermidis to a hydrophobic plastic surface. The assay is based upon the detection of cell-associated urease activity as a marker of bacteria remaining adherent to the polystyrene microwells of flat-bottomed, 96-well tissue culture plates. Using ATCC 35984, a slime-producing strain of S. epidermidis, the assay could detect as few as 3 x 10(3) bacteria and was linear to 3.5 x 10(7) bacteria. The adherence of both slime-positive and slime-negative coagulase-negative staphylococci could be evaluated by using this method. This assay could be used to examine factors which influence the adherence of individual S. epidermidis strains to hydrophobic surfaces and to develop agents or coating materials which suppress the adherence of coagulase-negative staphylococci to biomedical implants.

Efficacy and safety of mercuric oxide in the treatment of bacterial blepharitis.

A double-masked, placebo-controlled, randomized study was done to assess the safety and clinical and quantitative microbiologic efficacy of 1% mercuric oxide (yellow) ophthalmic ointment in the treatment of eyelid infections, i.e., bacterial blepharitis. A total of 39 patients with bacterial counts and clinical signs indicative of eyelid infection were treated twice daily for 7 days. Clinical biomicroscopic examination and quantitative microbiologic cultures were done just prior to initiation of treatment and again on days 3 and 7. Statistical analysis revealed a significant improvement in the clinical signs, bacterial count, cure rate, and improvement rate for subjects taking the active medication, compared with those taking the placebo on days 3 and 7. In addition, the medication was well tolerated.

Effect of preoperative fusidic acid on the normal eyelid and conjunctival bacterial flora.

A randomised trial comparing the topical application of 1% fusidic acid with 0.3% gentamicin solution in the reduction of the normal preoperative lid and conjunctival microbial flora was performed. Forty patients awaiting cataract surgery were randomly divided into two groups consisting of 20 patients each. The first group received a 1% microcrystalline suspension of fusidic acid, the second 0.3% gentamicin to the preoperative eye every two hours between 0600 and 2400 daily for 48 hours preoperatively. Cultures were obtained from both the lid margins and the conjunctival sac of both groups prior to antibiotic therapy and again in the operating theatre before surgery. Microbiological identification and colony counts were performed by standard laboratory methods. Staphylococcus epidermidis was the commonest micro-organism isolated. Statistical analysis revealed no significant differences in the ability of a 1% microcrystalline suspension of fusidic acid and 0.3% gentamicin in eliminating or reducing the normal preoperative conjunctival or lid flora.

Minocycline effects in patients with active trachoma.

Minocycline has a unique solubility in lipids and may reach therapeutic concentrations in tears and saliva. In two consecutive prospective double-masked clinical trials that were carried out in two villages in Saudi Arabia, we assessed the effects of oral minocycline in the treatment of trachoma and compared its effects with those of topical tetracycline ointment in the first study and to tetracycline ointment and placebo in the second study. A total of 178 eyes in 96 patients were included. The age range was 7 to 14 years, with a mean age of 9 years. All patients underwent complete ophthalmologic evaluation. School children were divided into two groups in a double-masked fashion. The first group received either oral minocycline or topical tetracycline 1% ointment and the second group was divided into three subgroups, each receiving one of the following therapeutic modalities: oral minocycline, topical tetracycline ointment, or placebo ointment. All patients were evaluated before initiation of therapy, at three weeks and at 12 months following treatment. Therapy was continued for a period of five weeks. These two double-masked field-based clinical trials have shown both minocycline given orally and tetracycline ointment given topically were effective in decreasing the intensity of inflammation due to trachoma. Oral minocycline was found to be equally effective as topical tetracycline ointment in the treatment of trachoma at three weeks. Minocycline, however, was found to be superior to topical tetracycline when patients were evaluated one year after therapy (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Monoclonal antibodies in the laboratory diagnosis of trachoma.

Various techniques which use monoclonal antibodies to detect Chlamydia trachomatis in clinical specimens are reviewed. An investigation comparing the efficacy of immunofluorescent staining with Giemsa staining in detecting Chlamydia in conjunctival scrapings from cases of active trachoma is presented. Sixty-two eyes of schoolboys with moderate to severe trachoma were studied. Giemsa staining detected chlamydial inclusion bodies in 34 percent of the specimens. Free elementary bodies were detected by fluorescent monoclonal antibody in 21 percent. Eleven percent were positive by both Giemsa and immunofluorescence and 55 percent were positive by either Giemsa and/or immunofluorescence. The addition of fluorescent monoclonal antibody assay to routine Giemsa staining resulted in an increase in the yield of positive specimens by 29 percent.