Virginia H. Morthland

Control of methicillin-resistant Staphylococcus aureus in a hospital and an intensive care unit.

OBJECTIVE To describe methicillin-resistant Staphylococcus aureus (MRSA) control in a hospital, including a surgical intensive care unit (SICU) outbreak. DESIGN Prospective surveillance of newly identified patients with MRSA. Barrier isolation (disposable gloves for direct contact with patient or immediate environment) was used for the routine care of hospitalized MRSA patients as of October 1991. Beginning in 1992, MRSA isolates were typed by restriction endonuclease enzyme analysis of plasmid DNA (REAP) and/or pulsed-field gel electrophoresis of genomic DNA (PFGE). Surveillance information and MRSA typing were used concurrently to identify nosocomial case clustering, confirm cross-infection, and support a need for additional outbreak control interventions. SETTING University-affiliated public hospital. PARTICIPANTS Patients with newly identified MRSA colonization or infection from 1991 through 1993 and epidemiologically associated staff providing care to eight SICU patients in an outbreak. INTERVENTIONS Barrier isolation for affected and unaffected patients in and admitted to the SICU institution when the outbreak was identified and cross-infection confirmed. Anterior nares cultures of staff in contact with outbreak cases for detection of MRSA colonization. RESULTS Fifty-six hospitalized patients with community-acquired MRSA and 80 patients with nosocomial MRSA colonization or infection were identified during the 3 years. After the introduction of barrier isolation, the annual frequency of new nosocomial MRSA cases decreased and only one outbreak (eight cases in the SICU) caused by type-related isolates occurred. The other 35 nosocomial cases of MRSA during 1992 and 1993 were not epidemiologically related or were caused by isolates with different types. The SICU outbreak ended after instituting barrier isolation for all patients (with and without MRSA) in and admitted to the unit. Six colonized SICU staff were identified. All outbreak cases had identical or related MRSA types by PFGE and REAP. Staff isolates were different from case isolates by typing, and staff were not restricted and not given treatment for colonization. After more than 6 months of follow up, no further outbreaks of MRSA in the SICU or elsewhere in the hospital occurred despite returning to barrier isolation for affected patients only. CONCLUSION MRSA in hospitals and outbreaks of MRSA in ICUs can be controlled by surveillance and minimal barrier interventions. REAP or PFGE typing of MRSA can be used to support or refute the presence of cross-transmission. Typing also may be helpful when planning and assessing the effectiveness of interventions directed at endemic, as well as outbreak, MRSA control.

The challenge of vancomycin-resistant enterococci: A clinical and epidemiologic study

BACKGROUND Vancomycin-resistant enterococci have been recovered with increasing frequency from hospitalized patients. Risk factors, mode of nosocomial transmission, extent of colonization in hospitalized patients, and treatment options for these organisms have not been completely delineated. METHODS We studied 53 patients (group A) with vancomycin-resistant enterococci isolated from various clinical specimens and also surveyed for vancomycin-resistant enterococci in stool specimens submitted for Clostridium difficile toxin assays (group B). Stool specimens submitted for identification of bacterial pathogens and stool specimens from hospital employees were also analyzed for vancomycin-resistant enterococci. RESULTS Seventy-six isolates of vancomycin-resistant enterococci were recovered in group A. Five of these patients harbored vancomycin-resistant enterococci on admission. Fifty-three of 289 group B stool specimens submitted for C. difficile toxin assays yielded vancomycin-resistant enterococci. Cephalosporins and vancomycin were the most common antimicrobial agents received by both groups of patients. Enterococcus faecium isolates were more resistant than Enterococcus faecalis isolates to antimicrobial agents. All isolates exhibited high-level aminoglycoside resistance and were not beta-lactamase producers. There were at least 15 different molecular clones of E. faecium and three of E. faecalis. Vancomycin-resistant enterococcal bacteremia was associated with a 100% in-hospital mortality rate. CONCLUSIONS Multidrug-resistant and vancomycin-resistant enterococci have become important nosocomial pathogens that are difficult to treat. Vancomycin-resistant enterococcal bacteremia was associated with a poor prognosis. We found a high rate of colonization in patients with suspected C. difficile toxin colitis. Judicious use of vancomycin and broad-spectrum antibiotics is recommended, and strict infection control measures must be implemented to prevent nosocomial transmission of these organisms.

Molecular detection of a common mutation in coagulation factor V causing thrombosis via hereditary resistance to activated protein C.

More than half of all patients with familial or recurring venous thrombosis have hereditary resistance to activated protein C (HRAPC) as the result of specific missense mutation in the gene for coagulation factor V. Because the mutant factor Va (with an Arg to Gln substitution at codon 506) cannot be cleaved and inactivated by activated protein C, carriers of this mutation are at significantly increased risk of venous thrombosis. We have recently introduced a direct polymerase chain reaction (PCR)-based clinical diagnostic test for the factor V codon 506 mutation based on the destruction of an Mnl I restriction site by the causative nucleotide substitution. To assess the accuracy of this PCR-based assay, we compared a functional clotting time test for HRAPC with the direct mutation test. Of 47 patients dually tested, only five had discrepant values for the functional test versus the DNA test. Either of these two complementary assays is useful for the accurate diagnosis of HRAPC. The DNA-based test is, however, specifically recommended for evaluation of anticoagulated patients or patients with borderline functional tests and confirmation of genotype in HRAPC families. In an additional analysis of 287 normal individuals, we found an extremely high prevalence of the mutated codon 506 allele-- approximately 4% in each of two different populations. The absence of disease in the majority of heterozygous carriers suggests that symptomatic thrombosis requires the simultaneous presence of both a mutated factor V protein and additional synergistic factors.

Plasmid DNA Fingerprinting of Acinetobacter calcoaceticus Subspecies anitratus From Intubated and Mechanically Ventilated Patients

Forty-three intubated and mechanically ventilated patients in five intensive care units (ICUs) of one hospital developed respiratory colonization or infection with Acinetobacter calcoaceticus subspecies anitratus over a 16-month interval. Neither the frequency nor rate of A anitratus isolation exceeded the hospital endemic norms. Single isolates from 34 of the patients were subtyped by plasmid DNA analysis, two biotyping systems and antimicrobial susceptibility to 24 drugs. Plasmid DNA fingerprints were distinct in 18 isolates (they differed from each other and all others), similar in two and identical or similar in ten. The latter group of isolates were recovered from patients in four ICUs. Reproducibility of biotyping was poor. Neither biotyping nor antimicrobial susceptibility were successful in identifying sameness among the group isolates nor differences among other isolates. We conclude that plasmid DNA fingerprinting should be used to assess the possibility of multiple patient transmissions of the same A anitratus strain in the absence of an obvious outbreak.

Effects of antacids and dialysate dwell times on multiple-dose pharmacokinetics of oral ciprofloxacin in patients on continuous ambulatory peritoneal dialysis.

Six stable patients on continuous ambulatory peritoneal dialysis were evaluated for the appearance of ciprofloxacin in their peritoneal dialysate following oral ingestion of 750 mg of the drug every 12 h for four doses. Three subjects participated in this study twice, once while taking and once while abstaining from phosphate-binding aluminum antacids. Subjects tolerated the medication without evidence of toxicity. Food may have delayed or decreased the absorption of ciprofloxacin, whereas antacids definitely decreased the absorption of the drug. Peak concentrations in serum noted in the absence of antacids ranged from 2.9 to 6.4 micrograms/ml, and peak concentrations in dialysate in the absence of antacids ranged from 1.8 to 4.5 micrograms/ml. Peak ciprofloxacin concentrations in serum achieved in subjects taking antacids were 14 to 50% of those achieved in subjects without antacids. The peak concentrations in dialysate achieved in subjects on antacids were 8 to 33% of those observed in subjects off antacids. The clearance of ciprofloxacin by continuous ambulatory peritoneal dialysis represented 2% of the total body (systemic) clearance. Simultaneous ratios of concentration in dialysate to concentration in serum (D/S) were determined at various durations of dialysate dwelling within the peritoneum. A progressive rise of the D/S ratio was noted as dwell time increased. At 4 h D/S was 0.57 +/- 0.07 (mean +/- standard error of the mean; n = 9), and at 8 h it was 0.75 +/- 0.04 (n = 26). Long-dwell exchanges may be necessary to achieve reasonable concentrations of orally ingested ciprofloxacin in dialysate.

Plasmid DNA analysis of Staphylococcus epidermidis isolated from blood and colonization cultures in very low birth weight neonates

We prospectively studied the course of colonization and sepsis with Staphylococcus epidermidis among 29 very low birth weight neonates undergoing prolonged umbilical catheterization. S. epidermidis bacteremia occurred in 7 patients. In 6 bacteremia was preceded by positive colonization cultures. Isolates obtained from nares, base of umbilicus, umbilical catheter entry sites, catheter tips and blood were examined for plasmid DNA profiles. In 4 patients the plasmid profiles of the catheter entry site isolates were identical with those of the blood isolates. In the other 3 bacteremic patients plasmid profiles of the catheter entry site and blood isolates were different. No correlation was observed in the plasmid DNA patterns of isolates obtained from catheter tip cultures as compared to the corresponding blood cultures. The blood isolates from bacteremic patients had different plasmid profiles.

Application of genomic DNA subtyping by pulsed field gel electrophoresis and restriction enzyme analysis of plasmid DNA to characterize methicillin-resistant Staphylococcus aureus from two nosocomial outbreaks

Pulsed-field gel electrophoresis (PFGE) and restriction enzyme analysis of plasmid DNA (REAP) were applied to study the epidemiologic relationship among methicillin-resistant Staphylococcus aureus (MRSA) isolates from outbreaks in two hospitals in São Paulo, Brazil: 82 MRSA isolates, 73 from a university hospital and nine from a general adult intensive care unit of a private hospital, were collected from 62 patients: 95% of the MRSAs were also resistant to gentamicin and ciprofloxacin. REAP subtyping of both collections identified six different subtypes: 55 (72.6%) MRSAs from the university hospital and nine isolates from the private hospital shared the same epidemic REAP subtype. Discrimination by restriction of genomic DNA with Sma I followed by PFGE enabled the identification of 14 DNA subtypes. Based on the combined REAP-genomic DNA subtype, the predominant subtype in the university hospital was A/A (44 isolates) whereas the epidemic subtype in the private hospital was A/M (seven isolates). The application of two typing methods showed better discrimination among MRSAs than did either method alone.

Molecular epidemiology of gastric colonization by Enterococcus faecalis in a surgical intensive care unit

We applied restriction endonuclease analysis of genomic DNA using pulsed field gel electrophoresis (PFGE) to study gastric colonization with Enterococcus faecalis among patients hospitalized in the surgical intensive care unit (SICU). Isolates were obtained by culturing prospectively the gastric contents of 140 patients in the SICU. In addition, cultures of respiratory specimens were obtained daily and cultures of blood, normally sterile body fluids, wounds, and urine were obtained when indicated clinically. A total of 177 isolates were obtained from 45 patients. Concentrations of E. faecalis in gastric fluid ranged from 1 x 10(2) colony forming units (CFU)/ml to greater than 5 x 10(7) CFU/ml (mean 8.0 x 10(6) CFU/ml). Overall, 33 different DNA types were identified by PEGE. In examining strain variation among isolates obtained from multiple anatomic sites over time, we found that the same DNA type was recovered from gastric aspirates, sputum, and wounds in a given patient and that these strains were carried over time. In general, given individuals were colonized with their own unique DNA type; however, one DNA type (type C) was shared by 11 different patients, and seven DNA types were shared by two individuals each. These results demonstrate the potential importance of gastric colonization as a reservoir for nosocomial strains of E. faecalis in an SICU setting.

Plasmid DNA analysis, biotyping, and antimicrobic susceptibility as subtyping tests for Klebsiella pneumoniae and Klebsiella oxytoca

We compared plasmid DNA analysis, biotyping by Vitek, and disk diffusion antimicrobic susceptibility as subtyping tests of Klebsiella pneumoniae and Klebsiella oxytoca. The 92 tested isolates were from alternate, culture-positive patients over 6 months. No outbreak or cluster of infections was recognized during this interval. Plasmid DNA was detected in 85% of the isolates. Each isolate except one had a reproducible absence of plasmid DNA or a reproducible plasmid DNA profile on repetitive testing. Restriction endonuclease enzyme analysis of plasmid DNA was necessary to distinguish differences among some isolates that had only large plasmids. Isolates with only large plasmids represented 18% of the collection. Of the 78 isolates with plasmid DNA, all but two were considered different from one another by plasmid DNA analysis. Biotyping and antimicrobic susceptibility testing were not highly reproducible. In addition, biotyping did not demonstrate a sufficient variety of patterns among the isolates for subtyping purposes. We conclude that plasmid DNA analysis is very useful as a subtyping test for isolates of K. pneumoniae and K. oxytoca. Neither biotyping nor antimicrobial susceptibility as performed in our laboratory had sufficient discriminatory power and reproducibility for subtyping these organisms.

Molecular epidemiology of Candida albicans colonization and fungemia in very low birthweight infants

OBJECTIVE This study investigated the relationship between colonization and fungemia. DESIGN This was a prospective study involving surveillance cultures of the nares, base of umbilicus, point of entry of umbilical catheter and parenteral fluids. Blood cultures were done when sepsis was suspected. All Candida albicans isolates were typed using restriction enzyme analysis of DNA. SETTING Patients were from the neonatal intensive care unit of a tertiary care hospital. POPULATION STUDIED Twenty-nine very low birthweight infants. MAIN RESULTS Eleven babies were colonized with C albicans and five of these babies developed fungemia, including five of seven who were colonized at the point of entry of the umbilical catheter. Three different strains of C albicans caused fungemia. In four of the five patients, initial catheter entry site isolates were identical to the subsequent blood isolates. Occasionally, infants were colonized with more than one strain of C albicans. CONCLUSIONS Preceding colonization with C albicans and, in particular, colonization at the site of entry of umbilical vascular catheters are risk factors for subsequent development of C albicans fungemia. Fungemic and colonizing isolates are usually identical to one another by DNA typing.