Indolent epidemic of Pseudomonas cepacia bacteremia and pseudobacteremia in an intensive care unit traced to a contaminated blood gas analyzer.

An epidemic of Pseudomonas cepacia bacteremia and pseudobacteremia occurred in the medical intensive care unit at the Clinical Center of the National Institutes of Health. Sixteen patients in the intensive care unit became colonized or infected with this organism in a 21-month period; whereas P. cepacia had been isolated only 16 times in the preceding 90 months from the entire hospital. Further analysis demonstrated a significant association of the epidemic cases with bloodstream isolation of the organism (p less than 0.001, Fisher's exact test). Mortality associated with bacteremia caused by P. cepacia was 38 percent. Intensive investigation of the intensive care unit and its surrounding environment eventually demonstrated that a blood gas analyzer in a satellite laboratory adjacent to the intensive care unit was the reservoir for the outbreak. Replacement of the machine resulted in termination of the outbreak, P. cepacia continues to represent an environmental threat to hospitalized patients.

Dysgonic fermenter 3-associated gastrointestinal disease in a patient with common variable hypogammaglobulinemia.

A gram-negative bacteria designated DF-3 was cultured on multiple occasions from stool samples of a patient with common variable hypogammaglobulinemia and chronic diarrhea. Antibiotic therapy resulted in elimination of the organism and resolution of the patient's symptoms. DF-3 has not been linked previously to human disease; because of its fastidious growth characteristics and unique isolation requirements, it may be a rarely identified cause of diarrhea and other gastrointestinal symptoms in immunocompromised patients.

Seven cases of surgical native valve endocarditis caused by coagulase-negative staphylococci: An underappreciated disease.

BACKGROUND: Native valve endocarditis caused by coagulase-negative staphylococci is uncommon and the diagnosis is infrequently considered. The disease, however, appears to be increasing in frequency and can pursue an aggressive clinical course. We report the clinical features of 7 cases of coagulase-negative staphylococcal native valve endocarditis (CNS-NVE) seen at 1 institution with a large cardiovascular referral base over a 10-month period. All cases required valve replacement surgery. METHODS: Clinical history, echocardiograms, and microbiologic and histopathologic data were reviewed for 7 patients with surgical CNS-NVE. RESULTS: Four patients had intravenous central catheters, and 1 had recent surgery, whereas the remaining 2 had no identifiable risk factors. Presentations ranged from subacute (4 cases) to acute with complications (3 cases). Complications included congestive heart failure, stroke, and heart block. Echocardiography demonstrated valvular lesions in all 7 cases. Valve pathologic study demonstrated gram-positive cocci in all 7 cases; blood cultures grew coagulase-negative staphylococci in 6 cases and valve cultures grew Staphylococcus epidermidis in 5 cases. CONCLUSIONS: Coagulase-negative staphylococci, including S epidermidis, can cause severe native valve endocarditis requiring valve replacement. The increasing use of intravascular access devices in the community may herald an increase in the incidence of CNS-NVE. A high index of diagnostic suspicion in the appropriate clinical setting is critical for optimal management.

Cytomegalovirus infection in children with human immunodeficiency virus infection.

OBJECTIVES: To determine retrospectively the prevalence of positive cytomegalovirus (CMV) cultures in pediatric patients with human immunodeficiency virus infection. METHODS: We reviewed the records of 273 children with human immunodeficiency virus infection referred to the Pediatric Branch of the National Cancer Institute for whom CMV cultures were performed between January, 1991, and October, 1994. RESULTS: Of this group 189 patients (69%) had negative CMV cultures and 84 (31%) had positive cultures. The prevalence of CMV-related disease was 9% for the entire group, including 4 (2.1%) patients with negative CMV cultures. Among the 84 patients with positive CMV cultures, 21 (25%) had evidence of CMV disease. Patients with positive CMV cultures had a statistically significant decrease in survival in the presence of severe immunocompromise defined as an age-corrected CD4 count of < 21%. Nine of 35 (26%) autopsies performed demonstrated evidence of CMV disease, including 7 patients with disseminated CMV disease. CONCLUSIONS: Although CMV disease appears to be less frequent in children than adults, CMV infection still contributes significantly to morbidity and mortality in this population, especially when combined with severe immunosuppression.

High-dose acyclovir and pre-emptive ganciclovir to prevent cytomegalovirus disease in myeloablative and non-myeloablative allogeneic stem cell transplantation.

We evaluated high-dose acyclovir and pre-emptive ganciclovir to prevent cytomegalovirus disease in myeloablative and non-myeloablative allogeneic stem cell transplantation. One hundred and seventy-four consecutive patients who were at risk for CMV infection (either recipient or donor seropositive) and received either intensive chemoradiotherapy and a T cell-depleted stem cell transplant followed by delayed add-back of donor T cells (TCDT: n = 98), or a non-myeloablative preparative regimen followed by an unmanipulated peripheral blood stem cell transplant (NMT: n = 76) from an HLA-identical sibling donor were studied. All received high-dose acyclovir (HDACV) from day - 7 for 3 months post-transplant in conjunction with weekly CMV pp65 antigenemia monitoring and pre-emptive treatment with intravenous immunoglobulin (not CMV-specific) and ganciclovir. The actuarial probabilities of developing pp65 antigenemia were 83 +/- 4% after TCDT and 41 +/- 6% after NMT (P < 0.00001) with reactivation occurring earlier in the TCDT group (the median 36 days vs 55 days). We observed no reactivation of CMV in seronegative recipients with a seropositive donor (n = 23). A total of 11 patients (5 in TCDT, 6 in NMT) developed CMV disease within 400 days after transplantation, and one death was clearly attributable to CMV interstitial pneumonitis (IP). This strategy was associated with effective control of CMV antigenemia in the majority of patients and near-complete eradication of fatal CMV IP.

Dental health and viridans streptococcal bacteremia in allogeneic hematopoietic stem cell transplant recipients.

Viridans streptococci were the most common cause of bacteremia in 61 consecutive myeloablative allogeneic hematopoietic stem cell transplant (HSCT) recipients, occurring in 19 of 31 bacteremic patients (61%) during the period of post-transplant neutropenia. Seven of the 19 had more than one viridans streptococcus in the same blood culture. Twenty isolates from 15 patients were Streptococcus mitis. Most viridans streptococci were resistant to norfloxacin, used routinely for prophylaxis. Comparison of the 19 patients with viridans streptococcal bacteremia with a contemporaneous group of 23 allogeneic HSCT recipients with fever and neutropenia but no identified focus of infection found that patients with viridans streptococcal bacteremia were more likely to have severe intraoral pathology while neutropenic (26% vs 0%) and slightly shorter interval between the last dental procedure and the onset of neutropenia (11 vs 14 days). Poor underlying dental health and the use of norfloxacin thus appear to predispose to viridans streptococcal bacteremia.

Medium for the simultaneous detection of pyocyanin and fluorescein pigments of Pseudomonas aeruginosa.

To help simplify the identification of Pseudomonas aeruginosa by clinical microbiology laboratories, the authors developed a new medium, pyocyanin-fluorescein agar (PFA), which enhances the production of both Pseudomonas pigments simultaneously. Production of pigments on PFA was equivalent to production on a pyocyanin agar (P agar) and a fluorescein agar (F agar) used in combination and was superior to either P agar or F agar used alone. The medium is simple to prepare and it detected pigment in 94% of P. aeruginosa isolates tested.

Detection of Legionella by PCR in respiratory specimens using a commercially available kit.

Using polymerase chain reaction (PCR) for the detection of pathogens that are difficult to grow, such as Legionella species, may reduce difficulties encountered with culture and immunofluorescent staining. We evaluated a commercial PCR and hybridization kit, designed for environmental samples, for the detection of Legionella in respiratory specimens. Sixteen Legionella species cultures tested positive with the Perkin Elmer Legionella EnviroAmp Amplification and Detection kits (Perkin Elmer, Foster City, Calif). The assay detected as few as 100 colony-forming units per milliliter of spiked bronchoalveolar lavage (BAL) fluid, and no false-negative results were obtained. PCR inhibition by blood in the specimens was removed by washing pelleted specimens in sterile distilled water. Of 126 specimens screened with the kit, 1 induced sputum and 3 BAL specimens were positive by PCR. All 4 were validated as true-positive results by culture or serologic testing. The entire PCR and hybridization assay can be completed in less than 6 hours, whereas isolation and identification by culture requires up to 12 days, and serologic conversion may not be demonstrated for weeks. Molecular techniques based on direct extraction and amplification of DNA from respiratory specimens nay be useful for the timely diagnosis of legionellosis.

Infections with Roseomonas gilardii and review of characteristics used for biochemical identification and molecular typing.

Roseomonas is a recently described genus of gram-negative coccobacilli formerly designated as "pink-coccoid" groups I through IV by the Centers for Disease Control and Prevention (Atlanta, Ga) because of the organism's characteristic pink colonies. Since 1991 we have isolated Roseomonas from eight patients; in seven from blood cultures and in one from a skin lesion. The seven blood isolates were from patients with clinically significant underlying diseases who had central venous catheters in place; the majority were associated with polymicrobial catheter infections. Additional characteristics of their infections are described. The eight isolates had originally been identified by us as Centers for Disease Control (CDC) pink-coccoid group III. These organisms were re-identified using the criteria of Rihs et al, and all isolates fit most closely with Roseomonas gilardii. Antibiotic profiles were fairly homogeneous showing susceptibility to many antibiotics, but uniform resistance to cefoxitin, ceftazidime, and piperacillin. Attempts to determine whether the isolates were the same strain by pulsed-field gel electrophoresis suggested that 3 of the isolates were similar. Random amplified polymorphic DNA analysis, however, demonstrated that each of the eight isolates was a unique strain.

Clinical evaluation of the BacT/Alert and isolator aerobic blood culture systems.

The BacT/Alert (BTA) (Organon Teknika, Durham, NC) and Isolator 10 (ISO) (Wampole Laboratories, Cranbury, NJ) blood culture systems were evaluated for their ability to detect aerobic and facultatively anaerobic microorganisms in blood of adult patients. For each culture 8 mL of blood was inoculated into both the aerobic standard BTA bottle and the ISO tube. Of 7,259 paired culture sets, 1,168 organisms were recovered, and 667 (57.1%) of these were considered clinically significant. This represented 540 clinically significant positive cultures from 266 patients. Of the significant isolates, 410 were recovered by both systems, 108 by BTA only and 149 by ISO only (P <.025). Overall, the BTA detected 77.7% of the significant isolates, whereas ISO detected 83.8%. The ISO recovered significantly more isolates of Staphylococcus aureus (P = .0001), coagulase-negative Staphylococcus spp (P <.01), and non-Enterobacteriaceae gram-negative rod species (P <.0025), whereas the BTA detected significantly more isolates of Streptococcus spp (P <.0025). Growth of S aureus (P <.0025), Enterococcus spp (P <.0025), and Streptococcus spp (P <.0075) was detected earlier by the BTA when laboratory coverage was available during the first shift only (7:30 AM to 4:00 PM), and additionally of Enterobacteriaceae (P <.0005) and other gram-negative rod species (P <.0001) if coverage was extended to 12:00 AM. Yeasts were detected more rapidly by the ISO (P <.0025). The ISO contamination rate (5.9%) was six times that of the BTA. Taking into account its ability to rapidly detect most organisms, its automated and thus labor-saving features, and the minimal contamination rate associated with its use, the BTA appears to be a reliable alternative to the ISO as a blood culturing system, although improvement in detection of staphylococci and non-Enterobacteriaceae gram-negative rods would be desirable.

Usefulness of pyridoxal-containing blood agar as a primary plating medium to enhance recovery of nutritionally deficient streptococci.

We evaluated the use of horse blood agar containing 0.0005% pyridoxal hydrochloride (HBAP) mainly for use as a primary plating medium for blood cultures, other sterile fluids, wounds, and abscesses in order to facilitate the isolation of nutritionally deficient streptococci (NDS). Our results showed that the addition of pyridoxal hydrochloride (pxl) permitted good growth of NDS within 24 h and did not inhibit or significantly alter the colony morphology of 34 different species of microorganisms and 296 isolates of beta hemolytic streptococci presumptively identified as Group A streptococci. The use of pxl-supplemented blood agar such as HBAP is recommended for primary plating as well as subculturing of blood cultures, and may also increase recovery of NDS from wounds and abscesses.

Detection of Mycobacterium avium-Mycobacterium intracellulare in blood cultures using concentrated and unconcentrated blood in conjunction with a radiometric detection system.

Studies were performed in order to determine optimal methods for isolating mycobacteria from blood. The combined use of a lysis-centrifugation (Isolator) system and a radiometric-detection system (Bactec) was compared to the use of either unconcentrated blood or centrifuged blood pellet in conjunction with the Bactec mycobacterial system. Results showed that for cultures with greater than 10 colony forming units (CFU) per ml of blood, the use of unconcentrated blood and centrifuged blood pellet was as sensitive as use of the Isolator concentrate. For cultures with lower colony counts (less than 10 CFU/ml), however, Isolator concentrate was consistently better than either centrifuged or unconcentrated blood. Since over half of our positive cultures had low colony counts, the use of Isolator concentrate inoculated into either a Bactec mycobacterial culture vial or onto Middlebrook agar slants is recommended over the use of either centrifuged blood pellet or unconcentrated blood inoculated into a Bactec mycobacterial culture vial.

Identification of clinical isolates of nondiphtherial Corynebacterium species and their antibiotic susceptibility patterns.

Starting in 1982, our laboratory has performed species identification of coryneform bacteria isolated from blood cultures, intravenous (i.v.) catheter tips and sites, urines with high colony counts, and other potentially significant cultures, using predefined criteria. Of 283 isolates identified, Corynebacterium jeikeium was the most common (47%), followed by CDC group G2 (12%) and C. minutissimum (8%). Blood cultures and i.v. catheter-related sources were the most frequent sources (58% of total). Certain species or groups, like CDC group G2, were most frequently isolated from blood or i.v. catheter sites. CDC group G2 showed a progression to greater multiple antibiotic resistance during this 9-year period. Occasional multiresistant strains of other species were also encountered. By in vitro testing, we note vancomycin remains the most active agent against corynebacterialike organisms, and is the most reliable antibiotic to use while awaiting susceptibility testing results.

Rapid identification of enterococci by pyrrolidonyl aminopeptidase activity (PYRase).

Group A streptococci and enterococci can be differentiated from other streptococci by their ability to cleave pyrrolidonyl beta-naphthylamide (PYRase). We evaluated two PYRase systems [Strep-A-Chek (SAC), E-Y Laboratories, San Mateo, CA; Strep-A-Fluor (SAF), BioSpec, Inc., Dublin, CA) for the presumptive identification of enterococci. Initially, 40 enterococcal and 21 nonenterococcal streptococci were tested, retrospectively. A prospective comparison of SAC and SAF to bile-esculin reaction (BE) was then incorporated into our routine procedure for the identification of non-beta-hemolytic streptococcal colonies from cultures. All isolates were speciated using standard biochemical tests. We encountered 85 enterococcal and 26 nonenterococcal isolates. Tests were performed on colonies from primary plates whenever possible (77 isolates). Sensitivity and specificity of both SAC and SAF were greater than 96% in identifying enterococci from routine cultures. These PYRase tests were cost-effective, easily adaptable to work-flow, and yielded results within 30 min. Thus, PYRase testing appears to be a reasonable alternative for the identification of enterococci in the clinical laboratory.

Development of a PCR assay for diagnosis of Pneumocystis carinii pneumonia based on amplification of the multicopy major surface glycoprotein gene family.

We have evaluated a PCR technique using primers based on Pneumocystis carinii major surface glycoprotein (MSG) genes, a multicopy gene family, for utility in detection of P. carinii in BAL and oropharyngeal samples obtained from immunosuppressed patients. These primers were able to detect P. carinii DNA in as little as 16 fg of genomic DNA. PCR using MSG primers detected P. carinii DNA in 7 smear-positive BAL samples (100% sensitivity), and found no P. carinii DNA in 12 smear-negative BAL samples (100% specificity). Mitochondrial ribosomal RNA (mrRNA) primers, commonly used in PCR studies of PCP, detected P. carinii in six of seven positive samples (85.7% sensitivity) and none of 12 were negative samples (100% specificity). Diagnosis of PCP by amplification of 81 oropharyngeal samples using MSG primers had a 50% sensitivity (4/8) and 96% specificity (70/73). PCR with mrRNA primers was 37.5% sensitive (3/8) and 100% specific (73/73). All three false-positive MSG results showed a very low intensity on Southern hybridization. PCR using MSG gene primers should prove valuable in the diagnosis of PCP.

Multicategory interpretive reporting of susceptibility testing with selected antimicrobial concentrations. Ten years of laboratory and clinical experience.

For the last 10 years, the NIH Microbiology Laboratory has been using a broth microdilution method to perform antibiotic susceptibility tests. Instead of using a continuous twofold dilution series as we had done for the previous 10 years, in 1978 we introduced a susceptibility panel that utilized a minimal number of selected (discontinuous) antimicrobic concentrations. The concentrations selected were those thought to be the most clinically relevant, based on known pharmacologic properties of each antimicrobic agent, as well as known MIC population distributions that we had acquired on 50,000 organisms in the preceding years. In addition to using selected concentrations, we also added interpretive codes to aid the physician in selecting the best antimicrobic agent to use. We had previously only reported quantitative MIC results without qualitative interpretations. The present interpretive criteria inform the physician not only if the organism is susceptible or resistant but also if intramuscular or intravenous doses are needed, if the organism is susceptible to an antimicrobic agent but only for lower urinary tract infections, if the organism is resistant to penicillin by virtue of penicillinase production, and in the case of streptococci, if streptomycin or gentamicin can be expected to show synergy when combined with a penicillin. The use of clinically relevant selected concentrations combined with clear interpretive criteria has worked well in our hospital setting. Physicians are able to understand and utilize the information effectively and have found almost no need for exact MICs using a twofold dilution series.

Nitroblue tetrazolium and Limulus assays for bacteremia after dental extraction: effect of topical antiseptics.

Bacteremia that occurs after dental extraction is common. This study assessed the effect of topical antisepsis on the incidence and magnitude of post-extraction bacteremia. On hundred patients scheduled for elective tooth extraction were randomized among four groups: contr-l, mouthrinsing with sodium-p-toluene sulfonchloramide (chloramine-T), toothbrushing with chloramine-T, and irrigation with Lugol's solution. The results showed that 84% of the control group and 59% of the treatment groups had positive blood cultures (290 organisms isolated) after dental extraction. The duration and magnitude of these bacteremias were diminutive as documented by the six serial blood cultures taken for each patient, colony counts per milliliter of blood, and nitroblue tetrazolium and Limulus assays. Brushing the teeth or rinsing the mouth with chloramine-T before dental extraction significantly reduced the incidence of bacteremia (P less than .025) and the number of different organisms recovered from each patient (P less than .05). Thus, topical treatment with chloramine-T is a simple and effective means of reducing the incidence of postextraction bacteremia.