Relevance of the number of positive bottles in determining clinical significance of coagulase-negative staphylococci in blood cultures.

Coagulase-negative staphylococci (CNS) are the most commonly isolated contaminants from blood cultures, yet they frequently cause true infections. Determining the clinical significance of CNS is difficult, and clinicians often consider the number of positive bottles within a set of blood culture bottles in their assessment. Therefore, in three separate studies, we counted the number of positive bottles within blood culture sets comprising two, three, or four bottles in order to predict whether or not CNS were clinically significant isolates (CSI) in adult patients with suspected sepsis. Each culture was evaluated by independent, published clinical criteria to determine its clinical importance. Of 486 positive sets that included two adequately filled bottles, 127 (26%) CNS were CSI, 329 (67%) were contaminants, and 30 (6%) were indeterminate as a cause of sepsis. Among CSI, 39 and 61% were isolated from one and two bottles, respectively. The positive predictive value for sepsis was 18% when one bottle was positive and 37% when both bottles were positive. Of 235 positive sets that included three adequately filled bottles, 81 (34%) were CSI, 109 (46%) were contaminants, and 45 (19%) were indeterminate as a cause of sepsis. Of CSI, 43, 38, and 19% were found in one, two, and three bottles, respectively. The positive predictive value for sepsis was 28, 52, and 30% when one, two and three bottles were positive. Of 303 positive blood culture sets that included four adequately filled bottles, 64 (21%) were considered CSI, 197 (65%) were contaminants, and 42 (14%) were indeterminate as a cause of sepsis. Of CSI, 27, 28, 19, and 27% were found in one, two, three, and four bottles, respectively. The positive predictive value for sepsis was 11, 30, 34, and 37% when one, two, three, and four bottles were positive. We conclude that the number of culture bottles positive in a given culture set cannot reliably predict the clinical significance of the CNS isolated and, therefore, should not be used as a criterion for determining whether or not an isolate represents true infection or contamination.

Human immunodeficiency virus type 1 drug resistance testing: a comparison of three sequence-based methods.

The use of genotypic assays for determining drug resistance in human immunodeficiency virus (HIV) type 1 (HIV-1)-infected patients is increasing. These tests lack standardization and validation. The aim of this study was to evaluate several tests used for the determination of HIV-1 drug resistance. Two genotypic tests, the Visible Genetics TruGene HIV-1 Genotyping Kit and the Applied Biosystems HIV Genotyping System, were compared using 22 clinical samples. Genotyping results were also obtained from an independent reference laboratory. The Visible Genetics and Applied Biosystems genotyping tests identified similar mutations when differences in the drug databases and reference strains were taken into account, and 19 of 21 samples were equivalent. The concordance between the two assays was 99% (249 of 252 mutation sites). Mutations identified by the reference laboratory varied the most among those identified by the three genotypic tests, possibly because of differences in the databases. The concordance of the reference laboratory results with the results of the other two assays was 80% (201 of 252). Samples with 500 to 750 HIV RNA copies/ml could be sequenced by the Visible Genetics and Applied Biosystems assays using 1 ml of input. The Visible Genetics and Applied Biosystems assays both generated an accurate sequence. However, the throughput of the Visible Genetics assay is more limited and may require additional instruments. The two assays differ technically but are similar in overall complexity. Data analysis in the two assays is straightforward, but only the reports provided by Visible Genetics contain information relating mutations to drug resistance. HIV drug resistance genotyping by sequencing is a complex technology which presents a challenge for analysis, interpretation, and reporting.

Controlled comparison of BacT/ALERT FAN aerobic medium and BATEC fungal blood culture medium for detection of fungemia.

Yeasts are an increasingly common cause of nosocomial bloodstream infections. Methods for their detection are many; controlled comparisons are few. The vented FAN aerobic blood culture medium has been shown to be superior to the standard BacT/ALERT aerobic medium for the detection of fungemia as well as bacteremia. The BACTEC selective fungal medium (FM) (BD Biosciences, Sparks, Md.) allowed detection of more episodes of fungemia than did a resin-containing medium with equal volumes of blood cultured. Therefore, we compared vented FAN to FM for the ability to recover fungi from the blood of patients who were at increased risk of having fungemia. From 5,109 cultures processed for which both FAN and FM bottles were adequately filled, fungi were recovered from 87 cultures. Of these, 47 were detected with both bottles, 12 were detected with FAN only, and 28 were detected with FM only (P < 0.05). FAN was the first bottle positive for 36 of the 47 cultures for which both bottles yielded the same fungus, whereas the FM bottle was the first bottle positive for 11 cultures (P < 0.001). A total of 54 episodes of fungemia were identified, with 40 detected by both media, 4 detected only by FAN, and 10 detected only by FM (P value, not significant). We conclude that the vented FAN aerobic bottle is comparable to the FM bottle for detection of episodes of yeast infection but has the added benefit of detecting bacteria.

Effect of indomethacin on peritoneal protein loss in a rabbit model of peritonitis.

BACKGROUND: Although various inflammatory mediators have been previously shown to be released into the peritoneal cavity during peritonitis in peritoneal dialysis patients, those that are involved in governing changes in peritoneal permeability to small solutes and protein remain incompletely defined. METHODS: We determined the importance of prostanoid production in the enhanced protein loss observed during acute peritonitis by inhibition experiments using indomethacin, an inhibitor of cyclooxygenase activity. The association between changes in peritoneal permeability and the generation of inflammatory mediators after adding Escherichia coli to peritoneal dialysate was first examined in series 1 experiments. Series 2 experiments then determined the effect of intraperitoneal administration of indomethacin (75 microg/mL) on changes in peritoneal permeability after adding E. coli to peritoneal dialysate. All experiments were performed in male New Zealand White rabbits (2.6 to 3.4 kg body weight) using an eight-hour dwell of dialysate containing 2.5% glucose. Peritoneal permeability to creatinine and protein was assessed by time-dependent changes in the dialysate to plasma concentration ratios of these solutes. RESULTS: Series 1 experiments showed enhanced leukocyte migration into the peritoneal cavity and increased peritoneal permeability to protein during bacterial challenge that was accompanied by an increase in the dialysate concentrations of prostaglandin E2 (PGE2), 6-keto-PGF1alpha, and interleukin-8, but not nitrate + nitrite (a measure of local nitric oxide production). Inhibition of prostanoid production by intraperitoneal administration of indomethacin in series 2 experiments resulted in lower dialysate concentrations of PGE2 and 6-keto-PGF1alpha and in lower peritoneal permeability to protein, both to control levels. No effect of indomethacin on transperitoneal migration of leukocytes or the generation of interleukin-8 was observed. CONCLUSIONS: Enhanced production of prostanoids likely plays an important role in governing the increase in peritoneal permeability to protein during acute, bacterial peritonitis in the rabbit.

Comparison of iodophor and alcohol pledgets with the Medi-Flex blood culture prep kit II for preventing contamination of blood cultures.

Iodophor and alcohol pledgets were compared with the Medi-Flex Prep Kit II for skin disinfection before venipuncture. Of 12,367 blood cultures collected, 6,362 were done with conventional pledgets and 6, 005 were done with Medi-Flex kits. Contamination occurred in 351 of 6,362 blood cultures (5.5%; range, 3.7 to 8.1%) with conventional pledgets versus 328 of 6,005 (5.5%; range, 3.5 to 7.5%) with Medi-Flex kits.

Community-acquired bacterial pneumonias.

Community-acquired bacterial pneumonias are among the most common of infectious diseases. The diagnosis of the etiological agent is becoming more challenging and more critical as new organisms are recognized as pathogens, and as well established agents become increasingly resistant to antimicrobial agents. The value of clinical microbiology laboratory data in the management of pneumonia is controversial. A well recognized, published guideline suggests that no laboratory studies be performed. Yet common practice and a more recent guideline advocate routine collection of sputum for Gram's stain and culture, along with traditional blood work. Given the increasing need to distinguish among a long list of possible pathogens and the need to recognize antibiotic resistance, it seems most prudent to include microbiological studies. This information can be used to guide initial therapy, and perhaps limit the overutilization of broad-spectrum antimicrobials. However, a prerequisite for the use of all cunently available test methods is their deployment in patients for whom there is clinical and radiographic evidence of pneumonia because recovery of a microorganism, especially from sputum, will occur with or without this clinical condition.

Increased protein loss during peritonitis associated with peritoneal dialysis is neutrophil dependent.

BACKGROUND: Peritonitis in peritoneal dialysis patients is accompanied by an enhanced migration of neutrophils (PMNs) and increased protein loss into the peritoneal cavity; however, the role of PMNs in governing increased protein loss during peritonitis associated with peritoneal dialysis is unknown. METHODS: We determined the importance of PMNs in governing changes in peritoneal permeability to protein in New Zealand White rabbits in which acute peritonitis was induced by adding 4 x 106 colony-forming units of Escherichia coli to 35 mL/kg of 0.9% saline dialysate. The total leukocyte and PMN migration into the peritoneal cavity was assessed by differential cell counts in the dialysate, and peritoneal permeability to protein was evaluated by calculating the dialysate to plasma concentration ratio for total protein as a function of time during a six- or eight-hour dwell. In series 1 experiments, leukocytes were depleted from the rabbit circulation by an intravenous injection of mustine (1.2 mg/kg) three days before the experiment; in series 2 experiments, integrin-dependent PMN migration into the peritoneal cavity was inhibited by an intravenous injection of monoclonal antibody (mAb) 60.3 (2 mg/kg) directed against the integrin CD18 on leukocytes five minutes before the experiment. RESULTS: In series 1 experiments, mustine decreased circulating leukocytes by 82 +/- 5% (mean +/- SEM) and circulating PMNs by 93 +/- 3%. Total leukocyte and PMN migration into the peritoneal cavity and peritoneal permeability to protein were decreased in mustine-treated rabbits after exposure to E. coli in the dialysate to levels similar to those found in rabbits without bacterial peritonitis. In series 2 experiments, an intravenous injection of anti-CD18 antibody also abrogated both the enhanced PMN migration into the peritoneal cavity and the increased peritoneal permeability to protein after exposure to E. coli in the dialysate. CONCLUSIONS: PMN migration into the peritoneal cavity is integrin dependent. Increased protein loss during acute, gram-negative bacterial peritonitis in a rabbit model of peritoneal dialysis is PMN dependent.

Role of the microbiology laboratory in the diagnosis of lower respiratory tract infections.

The appropriate use of the clinical microbiology laboratory for diagnosing lower respiratory tract infections is controversial. As in clinical care, it is crucial to categorize the presenting illness properly as acute bronchitis, an acute exacerbation of chronic bronchitis, community-acquired pneumonia, or nosocomial pneumonia if diagnostic efforts to establish a microbial etiology are to be productive for the individual patient and affordable to society. The greatest potential benefit of microbiological investigations lies in the etiologic diagnosis of pneumonia. For community-acquired pneumonia, evaluation of a gram-stained smear of sputum in terms of both quality and microorganisms present can help guide initial therapy as well as aid interpretation of subsequent culture results. As discussed in this review, the role of the clinical microbiology laboratory in the etiologic diagnosis of nosocomial and complicated pneumonias is more extensive and, in addition to evaluation of respiratory secretions, may include cultures of blood, pleural fluid, and specimens obtained by bronchoscopy. However, a prerequisite for the use of all currently available tests is their deployment for patients with clinical and radiographic evidence of pneumonia.

Disseminated mycobacterium avium complex infection presenting as osteomyelitis in a normal host.

Disseminated Mycobacterium avium complex (MAC) infection presenting as a painful lytic femur lesion with associated fever, night sweats and weight loss occurred in a 45-y-old woman with apparent normal immune function. Surgical drainage and 24 months of medical therapy resulted in a cure.

Update on detection of bacteremia and fungemia.

The presence of microorganisms in a patient's blood is a critical determinant of the severity of the patient's illness. Equally important, the laboratory isolation and identification of a microorganism present in blood determine the etiologic agent of infection, especially when the site of infection is localized and difficult to access. This review addresses the pathophysiology and clinical characteristics of bacteremia, fungemia, and sepsis; diagnostic strategies and critical factors in the detection of positive blood cultures; characteristics of manual and instrument approaches to bacteremia detection; approaches for isolating specific microorganisms associated with positive blood cultures; and rapid methods for the identification of microorganisms in blood cultures.

The clinical significance of positive blood cultures in the 1990s: a prospective comprehensive evaluation of the microbiology, epidemiology, and outcome of bacteremia and fungemia in adults.

To assess changes since the mid-1970s, we reviewed 843 episodes of positive blood cultures in 707 patients with septicemia. The five most common pathogens were Staphylococcus aureus, Escherichia coli, coagulase-negative staphylococci (CNS), Klebsiella pneumoniae, and Enterococcus species. Although CNS were isolated most often, only 12.4% were clinically significant. Half of all episodes were nosocomial, and a quarter had no recognized source. Leading identifiable sources included intravenous catheters, the respiratory and genitourinary tracts, and intraabdominal foci. Septicemia-associated mortality was 17.5%. Patients who received appropriate antimicrobial therapy throughout the course of infection had the lowest mortality (13.3%). Multivariate analysis showed that age (relative risk [RR], 1.80), microorganism (RR, 2.27), source of infection (RR, 2.86), predisposing factors (RR, 1.98), blood pressure (RR, 2.29), body temperature (RR, 2.04), and therapy (RR, 2.72) independently influenced outcome. Bloodstream infections in the 1990s are notable for the increased importance of CNS as both contaminants and pathogens, the proportionate increase in fungi and decrease in anaerobes as pathogens, the emergence of Mycobacterium avium complex as an important cause of bacteremia in patients with advanced human immunodeficiency virus infection, and the reduction in mortality associated with infection.

Clinical importance of increased sensitivity of BacT/Alert FAN aerobic and anaerobic blood culture bottles.

Two recent multicenter blood culture studies found that BacT/Alert FAN (FAN) bottles (Organon Teknika, Durham, N.C.) had increased yields in detecting bacteremia and fungemia compared with standard BacT/Alert (STD) bottles. Because the clinical importance of this increase in microbial recovery is unknown, we performed a retrospective analysis to determine the frequency with which FAN bottles were the sole means of detecting an episode of bacteremia. There were 1,047 positive blood cultures in which both study bottles were adequately filled and the organism isolated was judged to be the cause of sepsis: 240 (23%) were positive only in FAN bottles and 73 (7%) were positive only in STD bottles. Of a total of 664 episodes of bacteremia, 126 (19%) were identified only by FAN bottles and 43 (7%) were identified only by STD bottles (P < 0.0001). Episodes detected only by FAN bottles more often were recurrent events (23 of 126, or 18%) than episodes detected only by STD bottles (2 of 43, or 5%) (P < 0.05) and more commonly occurred in patients receiving theoretically effective antibiotic therapy (33 of 126 [26%] versus 4 of 43 [9%]) (P < 0.05). The medical records for patients with 127 of these episodes (92 FAN bottles only; 35 STD bottles only) were available for review. More than half of both FAN bottle-only (60 of 92, or 65%) and STD bottle-only (20 of 35, or 57%) episodes were judged to be clinically important. We conclude that FAN bottles improve the detection of bacteremia and that the majority of the additional episodes detected are clinically important. The benefits of the greater yield in specific patient populations must be balanced against the higher costs of FAN bottles.

Controlled evaluation of BacT/alert standard anaerobic and FAN anaerobic blood culture bottles for the detection of bacteremia and fungemia.

FAN medium was formulated to improve microbial recovery, particularly for fastidious microorganisms and for microorganisms causing sepsis in patients receiving antimicrobial therapy. In a controlled clinical evaluation performed at four university-affiliated hospitals, FAN anaerobic bottles were compared with standard anaerobic bottles for yield, speed of detection of microbial growth, and detection of septic episodes. A total of 10,431 blood culture sets were received; both anaerobic bottles of 7,694 blood culture sets were adequately filled with blood. Altogether, 925 isolates were recovered: 557 that were the cause of sepsis, 99 that were indeterminate as the cause of sepsis, and 269 contaminants. More Staphylococcus aureus (P < 0.001), coagulase-negative staphylococci (P < 0.001), Escherichia coli (P < 0.02), and all microorganisms combined (P < 0.005) were recovered from FAN bottles; more nonfermentative gram-negative bacilli (P < 0.05), Torulopsis glabrata (P < 0.001), and other yeasts (P < 0.01) were recovered from standard bottles. Growth of S. aureus (P < 0.001), coagulase-negative staphylococci (P < 0.001), Enterococcus faecalis (P < 0.025), streptococci other than Streptococcus pneumoniae (P < 0.01), and all microorganisms combined (P < 0.001) was detected earlier in standard bottles; growth of more isolates of E. coli (P < 0.05) and anaerobic bacteria (P < 0.01) was detected earlier in FAN bottles. The mean times to detection were 14.2 and 16.1 h for standard and FAN bottles, respectively. More septic episodes caused by S. aureus (P < 0.001), coagulase-negative staphylococci (P < 0.005), members of the family Enterobacteriaceae (P < 0.02), and all microorganisms combined (P < 0.02) were detected in FAN bottles; more septic episodes caused by nonfermentative gram-negative bacilli (P < 0.025) and yeasts (P < 0.005) were detected in standard bottles. In summary, more isolates (except for strict aerobes) were recovered from FAN bottles than from standard anaerobic bottles. Similarly, significant more septic episodes (except for those caused by strict aerobes) were detected with FAN bottles than with standard anaerobic bottles. With the exception of E. coli and anaerobic bacteria, growth of more isolates was detected earlier in standard anaerobic bottles.

Controlled evaluation of BacT/Alert standard aerobic and FAN aerobic blood culture bottles for detection of bacteremia and fungemia.

A new medium, FAN, designed to enhance the recovery of microorganisms, has been developed for the BacT/Alert blood culture system (Organon Teknika Corp., Durham, N.C.). We compared the yield and speed of detection of microorganisms in 6,847 adequately filled paired aerobic standard and FAN bottles at four university hospitals. Of 499 clinically significant microorganisms isolated from one or both bottles, significantly more Staphylococcus aureus isolates (P < 0.001), coagulase-negative staphylococci (P < 0.001), yeasts (P < 0.01), and all microorganisms combined (P < 0.001) were recovered from the FAN bottles. Overall, the speeds of detection of positive cultures did not differ between the two medium formulations; mean times to detection in the standard and FAN bottles were 16.1 and 16.0 h, respectively. When a subset of patients on antimicrobial therapy was evaluated, significantly enhanced yield from the FAN bottle was evident for staphylococci. Overall, the FAN bottle outperformed the standard aerobic BacT/Alert bottle.

Controlled evaluation of 5 versus 10 milliliters of blood cultured in aerobic BacT/Alert blood culture bottles.

Bottles developed for use in the BacT/Alert automated blood culture system (Organon Teknika Corp., Durham, N.C.) can accept up to 10 ml of blood without falling below a 1:5 ratio of blood to broth. We compared the yield and speed of detection of microorganisms in 13,128 adequately filled, paired, aerobic bottles inoculated with 5 versus 10 ml of blood at three university hospitals. A total of 798 microorganisms causing sepsis grew in one or both bottles. The overall recovery of microorganisms from 10-ml samples exceeded that from 5-ml samples (P < 0.001); the increased yield attributed to the additional 5 ml in the samples was 7.2%. The increased yield from 10-ml inocula was most marked for Escherichia coli (P < 0.01) and other members of the family Enterobacteriaceae (P < 0.001). Ten-milliliter samples did not yield more gram-positive bacteria, nonfermentative gram-negative rods, or yeasts. When both bottles were positive, the bottles inoculated with 10 ml of blood showed growth sooner (P < 0.001). Earlier detection with 10-ml inocula was especially notable for coagulase-negative staphylococci (P < 0.001), streptococci (P < 0.001), E. coli (P < 0.025), and other members of the family Enterobacteriaceae (P < 0.025). We conclude that an increase in the volume of blood inoculated into BacT/Alert aerobic blood culture bottles from 5 to 10 ml will increase the overall yield and the speed of detection of clinically important blood pathogens.

Catheter-related infections and blood cultures.

Catheter-related infections with both local inflammation and bacteremia or fungemia are common in hospitalized patients. The diagnosis of these infections is not, however, straightforward. Evidence of local inflammation is helpful, if present, but is not always found with site infections, and blood cultures are not positive. Systemic infection is associated with positive blood cultures, but the finding of a positive blood culture does not identify the catheter as the source. With central catheters, making a diagnosis without having to remove the catheter would be useful, because many of these patients could be treated with antibiotics without catheter removal. Multiple methods have been described for identification of these infections. Semiquantitative cultures of the catheter tip performed by rolling the catheter on the surface of an agar plate are the most popular. For central catheters, many advocate obtaining blood cultures through the catheter and comparing the results by quantitative methods with peripherally obtained blood cultures. No method has clearly demonstrated a clinical benefit in large numbers of patients. Because the most serious manifestation of catheter-related infection is bacteremia or fungemia, ordinary blood cultures are of the most practical importance in the identification of patients requiring therapy. Whether any of the additional studies described can be justified in everyday laboratory practice or simply represent considerable wasted effort is not known. Better methods for identifying infections and for managing such infections in patients with long-term indwelling central catheters are needed.

Laboratory detection of mycobacteremia.

Mycobacteremia, rarely seen or recognized before the AIDS epidemic, has now become common in patients with advanced HIV infection. With the introduction of new media and techniques for preparing specimens for media inoculation that lyse and concentrate organisms, our ability to detect mycobacteremia has improved such that we also occasionally detect these organisms in the blood of individuals with other underlying conditions. A precise understanding of the clinical and epidemiologic manifestations of mycobacteremia is not yet available, however, and additional advancements in blood culture techniques and alternative approaches to diagnosis will continue to be important in clinical microbiological research.

Q fever.

Q fever is an acute febrile illness first described in 1935 and now seen in many parts of the world. Human infection follows exposure to animals, especially domestic livestock. Recent outbreaks in metropolitan areas have implicated cats as the carrier of disease to humans. The etiologic agent, Coxiella burnetti, belongs to the family Rickettsiaceae, although it has distinct genetic characteristics and modes of transmission. Most recent attention has been focused on a number of large outbreaks of Q fever associated with medical research involving pregnant sheep. Although most infections are self-limited, some patients require prolonged treatment. Recent vaccines have had encouraging success in the prevention of disease in individuals at high risk of exposure.

Recovery of clinically important microorganisms from the BacT/Alert blood culture system does not require testing for seven days.

Recently, we published a comparison of the BacT/Alert blood culture system with the BACTEC 660/730 nonradiometric blood culture system using blood inocula of 5 ml per bottle. By reanalyzing data collected during that study, we found that, for true-positive isolates causing bacteremia or fungemia, 363 (97.6%) of 376 and 341 (97.7%) of 349 isolates were recovered by the end of day 5 of testing, and 364 (97.9%) of 376 and 343 (98.3%) of 349 isolates were recovered by the end of day 6 of testing for aerobic and anaerobic bottles, respectively. Most isolates recovered on days 6 (24 of 27) and 7 (20 of 25) of testing were either contaminants or indeterminate as a cause of sepsis. When used as recommended by the manufacturer, only six (1.3%) of 464 clinically important isolates recovered on test days 6-7 would have gone undetected had testing been limited to 5 days and four (0.9%) of 464 had testing been limited to 6 days. We conclude that BacT/Alert bottles can be tested for as few as 5 days and then discarded with minimal loss of true-positive isolates and maximal reduction of contaminants.