Multicenter Evaluation of BD Max Enteric Parasite Real-Time PCR Assay for Detection of Giardia duodenalis, Cryptosporidium hominis, Cryptosporidium parvum, and Entamoeba histolytica.

Common causes of chronic diarrhea among travelers worldwide include protozoan parasites. The majority of parasitic infections are caused by Giardia duodenalis, Entamoeba histolytica, Cryptosporidium parvum, and Cryptosporidium hominis Similarly, these species cause the majority of parasitic diarrhea acquired in the United States. Detection of parasites by gold standard microscopic methods is time-consuming and requires considerable expertise; enzyme immunoassays and direct fluorescent-antibody (DFA) stains have lowered hands-on time for testing, but improvements in sensitivity and technical time may be possible with a PCR assay. We performed a clinical evaluation of a multiplex PCR panel, the enteric parasite panel (EPP), for the detection of these common parasites using the BD Max instrument, which performs automated extraction and amplification. A total of 2,495 compliant specimens were enrolled, including 2,104 (84%) specimens collected prospectively and 391 (16%) specimens collected retrospectively. Approximately equal numbers were received in 10% formalin (1,273 specimens) and unpreserved (1,222 specimens). The results from the EPP were compared to those from alternate PCR and bidirectional sequencing (APCR), as well as DFA (G. duodenalis and C. parvum or C. hominis) or trichrome stain (E. histolytica). The sensitivity and specificity for prospective and retrospective specimens combined were 98.2% and 99.5% for G. duodenalis, 95.5% and 99.6 for C. parvum or C. hominis, and 100% and 100% for E. histolytica, respectively. The performance of the FDA-approved BD Max EPP compared well to the reference methods and may be an appropriate substitute for microscopic examination or immunoassays.

Multicenter evaluation of the BD max enteric bacterial panel PCR assay for rapid detection of Salmonella spp., Shigella spp., Campylobacter spp. (C. jejuni and C. coli), and Shiga toxin 1 and 2 genes.

Diarrhea due to enteric bacterial pathogens causes significant morbidity and mortality in the United States and worldwide. However, bacterial pathogens may be infrequently identified. Currently, culture and enzyme immunoassays (EIAs) are the primary methods used by clinical laboratories to detect enteric bacterial pathogens. We conducted a multicenter evaluation of the BD Max enteric bacterial panel (EBP) PCR assay in comparison to culture for the detection of Salmonella spp., Shigella spp., Campylobacter jejuni, and Campylobacter coli and an EIA for Shiga toxins 1 and 2. A total of 4,242 preserved or unpreserved stool specimens, including 3,457 specimens collected prospectively and 785 frozen, retrospective samples, were evaluated. Compared to culture or EIA, the positive percent agreement (PPA) and negative percent agreement (NPA) values for the BD Max EBP assay for all specimens combined were as follows: 97.1% and 99.2% for Salmonella spp., 99.1% and 99.7% for Shigella spp., 97.2% and 98.4% for C. jejuni and C. coli, and 97.4% and 99.3% for Shiga toxins, respectively. Discrepant results for prospective samples were resolved with alternate PCR assays and bidirectional sequencing of amplicons. Following discrepant analysis, PPA and NPA values were as follows: 97.3% and 99.8% for Salmonella spp., 99.2% and 100% for Shigella spp., 97.5% and 99.0% for C. jejuni and C. coli, and 100% and 99.7% for Shiga toxins, respectively. No differences in detection were observed for samples preserved in Cary-Blair medium and unpreserved samples. In this large, multicenter study, the BD Max EBP assay showed superior sensitivity compared to conventional methods and excellent specificity for the detection of enteric bacterial pathogens in stool specimens.

Pediatric multicenter evaluation of the Verigene gram-negative blood culture test for rapid detection of inpatient bacteremia involving gram-negative organisms, extended-spectrum beta-lactamases, and carbapenemases.

We evaluated the investigational use only (IUO) version of the rapid Verigene Gram-negative blood culture test (BC-GN), a microarray that detects 9 genus/species targets (Acinetobacter spp., Citrobacter spp., Enterobacter spp., Escherichia coli/Shigella spp., Klebsiella oxytoca, Klebsiella pneumoniae, Proteus spp., Pseudomonas aeruginosa, and Serratia marcescens) and 6 antimicrobial resistance determinants (blaCTX-M, blaKPC, blaNDM, blaVIM, blaIMP, and blaOXA) directly from positive blood cultures. BC-GN was performed on positive BacT/Alert Pediatric FAN and Bactec Peds Plus blood cultures with Gram-negative organisms at two tertiary pediatric centers. Vitek MS (bioMérieux, Durham, NC) was used to assign gold standard organism identification. The Check MDR CT-102 microarray (Check Points B.V., Wageningen, Netherlands) was used as an alternative method for detecting resistance determinants. In total, 104 organisms were isolated from 97 clinical blood cultures. BC-GN correctly detected 26/26 cultures with Acinetobacter spp., P. aeruginosa, and S. marcescens, 5/6 with Citrobacter spp., 13/14 with Enterobacter spp., 23/24 with E. coli, 2/3 with K. oxytoca, 16/17 with K. pneumoniae, and 0/1 with Proteus spp. BC-GN appropriately reported negative BC-GN results in 8/13 blood cultures that grew organisms that were not represented on the microarray but failed to detect targets in 3/5 cultures that grew multiple Gram-negative organisms. BC-GN detected 5/5 and 1/1 clinical blood cultures with blaCTX-M and blaVIM. All 6 results were corroborated by Check MDR CT-102 microarray testing. The Verigene BC-GN test has the potential to expedite therapeutic decision making in pediatric patients with Gram-negative bacteremia. Sensitivity was satisfactory but may be suboptimal in mixed Gram-negative blood cultures.

Multilab evaluation of delafloxacin MIC Test Strip against Gram-negative and Gram-positive organisms.

The performance of the delafloxacin MIC Test Strip (MTS) was evaluated. Three testing sites collected/tested clinical isolates, and 1 site tested challenge isolates that together total 224 S. aureus, 36 S. haemolyticus, 23 S. lugdunensis, 105 E. faecalis, 308 Enterobacteriales, and 140 P. aeruginosa. MIC testing was performed by broth microdilution (BMD) and MTS. Each site also tested 20 common isolates in triplicate on 3 days by MTS and 20 replicates of 4 QC strains by MTS and BMD. MTS results for consolidated clinical/challenge isolates were within 1 doubling dilution of the BMD MIC for 96.9% of S. aureus; 100% of S. haemolyticus, S. lugdunensis, and E. faecalis; 98.4% of Enterobacteriales; and 97.9% of P. aeruginosa. All reproducibility results were within 1 dilution of the modal MIC. All BMD and MTS results for the QC strains were within expected ranges. Overall, the delafloxacin MTS performed similar to BMD.

Quantitative skin cultures at potential catheter sites in neonates.

OBJECTIVE: To identify and quantify the bacterial and fungal flora present at body sites used for vascular catheterization of infants in a neonatal intensive care unit. DESIGN: Quantitative skin cultures were obtained from a group of neonatal patients to determine the bacterial flora found on the skin at four sites. Quantitative cultures of the jugular, subclavian, umbilical, and femoral sites were obtained on 50 infants, ranging in age from 2 days to 3 months old. SETTING: The neonatal intensive care unit of St. Christopher's Hospital for Children, a university-affiliated tertiary care children's hospital. RESULTS: Colony counts ranged from 0 to 10(6) colony-forming units/10 cm2. Types of organisms found were consistent with other published studies and included coagulase-negative staphylococci, Staphylococcus aureus, yeast, aerobic gram-negative rods, Enterococcus species, Corynebacterium species, and alpha-hemolytic streptococci. There was a significantly higher mean colony count at the combined jugular/femoral sites versus the subclavian site (P < 0.01) and umbilical site (P < 0.05). Mean colony counts did not differ significantly between the jugular and femoral site, or between the subclavian and umbilical site. The umbilical site was more likely to be colonized with aerobic gram-negative rods, Enterococcus species, and yeast, while the subclavian had coagulase-negative staphylococci as the predominant organism. The jugular and femoral sites demonstrated a higher colony count of aerobic gram-negative rods, Enterococcus species and yeast than the other sites. If central venous catheters need to be in place for extended periods of time, placement at a site with lower bacterial densities on the skin may help minimize catheter-associated infections. This study supports the subclavian as the preferred site.

Recovery of Pseudomonas cepacia and other Pseudomonas species from the environment.

Nine hundred sixteen cultures were obtained from homes of patients with cystic fibrosis, control homes, salad bars, and food markets, and analyzed for the presence of Pseudomonas cepacia and related bacteria. P cepacia was recovered from 5 (18%) of 27 homes, and from 20 (4%) of 509 cultures collected outside of homes. Relative to other pseudomonads, P cepacia is found infrequently in the environment. It is not clear how frequently these sources contribute to acquisition of this bacteria by persons with cystic fibrosis.

Comparative in vitro activity of furopenem against aerobic bacteria isolated from pediatric patients.

The in vitro activity of the oral penem furopenem (WY-49605, 545555, SUN5555, and ALP201) was tested against clinical bacteria isolated from pediatric patients. Furopenem was compared with clarithromycin, cefpodoxime, amoxicillin, amoxicillin-clavulanate, cefaclor, cefixime, and cefuroxime. Furopenem demonstrated consistent activity against Escherichia coli [minimum inhibitory concentration (MIC90) = 1.0 microgram/ml)] Klebsiella pneumoniae (MIC90 = 2.0 micrograms/ml), Salmonella enteriditis and Shigella spp. (MIC90 = 1.0 microgram/ml), and beta-lactamase-positive or -negative Haemophilus influenzae (MIC90 = 1.0 microgram/ml) and Moraella catarrhalis (MIC90 = 1.0 microgram/ml). Furopenem was also active against a number of the Gram-positive organisms tested including methicillin-susceptible Staphylococcus aureus and penicillin-susceptible Streptococcus pneumoniae. These results suggest a potential application for this agent in the treatment of children as outpatients.

The bactericidal activity of ampicillin, daptomycin, and vancomycin against ampicillin-resistant Enterococcus faecium.

Ampicillin, daptomycin, and vancomycin, alone and in combination with gentamicin, were examined for bactericidal effects on ampicillin-resistant Enterococcus faecium using broth dilution minimum inhibitory concentrations (MICs) and time-kill studies. We tested 12 ampicillin-resistant isolates and demonstrated the following MICs and MBCs, respectively: ampicillin, greater than or equal to 32 micrograms/ml and greater than 256 micrograms/ml; daptomycin, less than or equal to 4 micrograms/ml and less than or equal to 16 micrograms/ml; and vancomycin, less than or equal to 4 micrograms/ml and greater than 64 micrograms/ml. Time-kill studies demonstrated that daptomycin alone had marked activity against the ampicillin-resistant E. faecium and that the addition of gentamicin resulted in synergistic killing. In addition, ampicillin and vancomycin were not bactericidal for the ampicillin-resistant isolates without the addition of gentamicin. The present study supports the consideration of daptomycin alone or in combination with an aminoglycoside as an alternative therapy for ampicillin-resistant enterococci, although additional clinical experience is now necessary.

Comparison of susceptibility test methods to detect penicillin-resistant Streptococcus pneumoniae.

The detection of penicillin-resistant Streptococcus pneumoniae was assessed by six different methods: agar dilution, oxacillin screen by disk diffusion, E-test, and three overnight microdilution test methods that included commercial panels from MicroScan and Micro Media and in-house-made conventional panels using a commercial Haemophilus test medium (HTM) broth. Of the 52 pneumococcal isolates tested, 12 were resistant, 16 were relatively resistant, and 24 were susceptible to penicillin as defined by the reference agar dilution method. The oxacillin screen detected as resistant all 28 resistant and relatively resistant strains. The percentage of penicillin-resistant isolates detected by each minimum inhibitory concentration (MIC) test method was as follows: E-test (100%), Micro Media (75%), MicroScan (0%), and HTM (0%). With the relatively resistant isolates, the detection percentage was as follows: E-test (88%), Micro Media (94%), MicroScan (69%), and HTM (69%). In conclusion, the E-test and Micro Media MIC tests are acceptable confirmatory tests for detecting penicillin resistance among S. pneumoniae isolates.

Susceptibility testing of methicillin-resistant Staphylococcus aureus with three commercial microdilution systems.

The antimicrobial susceptibilities of 100 methicillin-resistant Staphylococcus aureus isolates were determined concurrently by API Uniscept KB, Micro-Media, MicroScan, standardized disk diffusion, and reference broth microdilution to evaluate whether these commercial microdilution systems would reliably defect methicillin-resistant S. aureus. The methicillin minimal inhibitory concentration for all isolates was greater than or equal to 16 micrograms/ml as determined by the reference minimal inhibitory concentration panels containing 2% NaCl supplemented Mueller-Hinton broth. Using the breakpoints established by the National Committee for Clinical Laboratory Standards for reporting susceptible and resistant methicillin results, there was 100% agreement between the reference methods and API Uniscept KB at 24 hr. The Micro-Media and MicroScan systems had 47% and 8% very major discrepancies at 24 hr, respectively. At 48 hr, these two systems exhibited 15% and 0% very major discrepancies. Micro-Media and MicroScan were in agreement with the reference microdilution method (+/- 1 log2 dilution) for 62% and 68% of the strains at 24 hr, respectively and 88% and 85% of the isolates at 48 hr, respectively. The results of this study indicate that API Uniscept KB would provide a practical and reliable method for the detection of methicillin-resistant S. aureus.

Mycobacterium fortuitum peritonitis in a patient undergoing chronic peritoneal dialysis.

Peritonitis, due to Mycobacterium fortuitum, developed in a 15-yr-old young man undergoing chronic peritoneal dialysis. Although of low pathogenic potential, this rapidly growing non-tuberculous mycobacterium does cause human disease particularly in the compromised host and should be considered as a potential cause of peritonitis in the chronic peritoneal dialysis patient.

Susceptibility testing of fastidious organisms in the API microdilution panel.

It has become increasingly important to perform routine susceptibility tests on certain anaerobic bacteria, Haemophilus influenzae and Streptococcus pneumoniae, as a result of the decreasing predictability of their antimicrobial susceptibility patterns. The antimicrobial susceptibilities of 49 anaerobic isolates, 25 H. influenzae isolates, and 25 S. pneumoniae isolates were determined concurrently by API Uniscept MIC trays and a conventional broth microdilution method using Wilkins-Chalgren broth, 5% Fildes in Schaedler broth, or 5% lysed horse blood in Mueller-Hinton broth, respectively. Analysis of 490 anaerobic organism-antibiotic combinations, 144 H. influenzae-antibiotic combinations, and 125 S. pneumoniae-antibiotic combinations showed that 98.9%, 100%, and 99.2%, respectively, of the API results were within +/- 1 log2 dilution of the reference system. The API Uniscept MIC panel would be acceptable for use as a routine susceptibility system for anaerobic organisms in a clinical microbiology laboratory. To eliminate trailing endpoints, however, further studies need to be performed to evaluate additional broth media for the susceptibility testing of H. influenzae and S. pneumoniae in the API panels.

In vitro activity of oral antimicrobial agents against clinical isolates of Pasteurella multocida.

Pasteurella multocida causes a wide variety of infections and is the most common localized soft tissue infection after animal bite injuries. Penicillin or amoxicillin has been considered agent of choice for therapy. Reported beta-lactamase production by some isolates, the therapeutic dilemma of the penicillin allergic patient, and the polymicrobial nature of some infections led to this study of alternate antimicrobial agents. The in vitro activity of ampicillin, amoxicillin/clavulanate, cefprozil, cefuroxime, erythromycin, clarithromycin, trimethoprim/sulfamethoxazole, ciprofloxacin, and tetracycline were compared to penicillin against 73 geographically diverse isolates of P. multocida from human infections collected since 1991. MIC90 (microgram/mL) were as follows: penicillin < or = 0.06; ampicillin < or = 0.5; amoxicillin/clavulanate < or = 0.5; cefaclor 1.0; cefprozil 1.0; cefpodoxime 0.06; cephalothin 2.5; cefuroxime < or = 0.25; erythromycin 2.0; azithromycin 1.0; clarithromycin 4.0; trimethoprim/sulfamethoxazole < or = 0.5/9.5; ciprofloxacin < or 0.25; tetracycline < or = 2.0. No beta-lactamase producing isolates were found in this study. This in vitro study has identified alternate oral agents to penicillins that may be appropriate for therapy of P. multocida infections.

Comparison of five different susceptibility test methods for detecting antimicrobial agent resistance among Haemophilus influenzae isolates.

The detection of antimicrobial agent resistance among ninety-eight Haemophilus influenzae isolates was assessed by six different antibiotic test methods: agar dilution on Mueller-Hinton agar supplemented with 5% lysed horse blood (MH-LHB), E-test using both Haemophilus test medium (HTM) agar and chocolate Mueller-Hinton (CMH) agar plates, Vitek Haemophilus susceptibility cards, and three overnight microdilution systems that included two commercial systems, Micro-Media and MicroScan, and the reference broth microdilution method using HTM broth. Agents tested in the study included ampicillin, amoxicillin/clavulanic acid (A/C), cefaclor, cefuroxime, cefotaxime, ceftriaxone, chloramphenicol, and trimethoprim/sulfamethoxazole. Both the reference HTM microbroth dilution method and agar dilution correctly classified all nine of the beta-lactamase negative ampicillin resistant (BLNAR) isolates. Each of the other test methods failed to detect one of the BLNAR strains, either because of growth failure (Micro-Media and MicroScan) or miscategorization of an isolate as susceptible (E-Test HTM, E-Test CMH, and Vitek). None of the test methods detected all six isolates identified as A/C resistant by HTM microbroth dilution. Of the remaining antimicrobials tested, ampicillin and cefuroxime yielded data that could be compared by all test methods. The very major, major, and minor errors for these two antimicrobials in comparison to the reference HTM microdilution method were as follows: Micro-Media (1.7%, 0%, and 4.8%); MicroScan (11.9%, 0%, and 8.1%); E-Test HTM (1.6%, 0%, and 2.0%); E-Test CMH (1.6%, 1.6%, and 4.6%); Vitek (8.1%, 0%, and 3.1%); and agar dilution on MH-LHB (0%, 0%, and 4.6%). Micro-Media and MicroScan panels failed to support the growth of 4.1% and 5.1% of the isolates, respectively.

Peritonitis involving Capnocytophaga ochracea.

Spontaneous bacterial peritonitis, due to Capnocytophaga ochracea, developed in a 65-year-old alcoholic patient with extensive cirrhosis and ascites. Previously reported human infections with this organism have included peridontal diseases, septicemia, and arthritis. This is the first report of spontaneous bacterial peritonitis associated with Capnocytophaga species.

The growth and survivability of Streptococcus pneumoniae clinical isolates subjected to various environmental conditions.

Historically, it has been hypothesized that environmental stress would favor the survival of antibiotic susceptible bacteria over resistant ones; however, there is little direct evidence to support this theory. Clinical isolates of S. pneumoniae were chosen and categorized as: penicillin susceptible, quinolone susceptible (PSQS, n = 3); penicillin resistant, quinolone susceptible (PRQS, n = 3); and penicillin resistant, quinolone resistant (PRQR, n = 5). Baseline growth of each isolate was measured by optical density for 24 h. The resulting optical density curves were compared to those obtained for the same isolates subjected to changes in environmental conditions, such as various temperature, pH, and diluted media. In addition, each isolate was inoculated onto cotton fiber disks, held at room temperature, and the recoverable CFU measured over 144 h. In comparison to controls grown under ideal conditions, the density of PSQS isolates was significantly lower than PRQR isolates after 24 h for the following conditions (p < 0.01): incubation at 40 degrees C (1.3 log10 lower); at pH 6.5 (1.6 log10 lower); and in limited nutrient conditions (1.36 log10 lower). When inoculated onto cotton fiber disks, the PRQR isolates decreased an average of 5.0 log10 after 72 h as compared to controls. In contrast, PSQS isolates decreased an average of 8.1 log10 (p < 0.01). Results of this study support the concept that antibiotic resistant isolates may not be at a competitive disadvantage in comparison to susceptible isolates when subjected to some adverse environmental conditions.

Antimicrobial susceptibility of clinical isolates of Brucella.

A number of antimicrobial agents have been used in the treatment of human brucellosis with varying effectiveness. The purpose of this study was to test the in vitro susceptibility of isolates of four Brucella species to a variety of antimicrobial agents, and to study in vitro synergy of combinations of agents. Minimal inhibitory concentrations (MICs) were determined using conventional broth microdilution methods and commercially available systems. Conventional checkerboard synergy microdilutions were prepared for gentamicin or streptomycin plus tetracycline, and rifampicin plus tetracycline. Synergy or antagonism was determined by the fractional inhibitory concentration index. Penicillin G and ampicillin showed in vitro activity against Brucella (MIC90 4 micrograms/ml), whereas the antipseudomonal penicillins were less active (carbenicillin MIC90 12 micrograms/ml, piperacillin MIC90 32 micrograms/ml). Among the third generation cephalosporins tested, cefotaxime (MIC90 2 micrograms/ml) demonstrated greatest activity. As a class, aminoglycosides were equivalent (MIC90 1-4 micrograms/ml). All strains were sensitive to tetracycline (MIC90 0.25 microgram/ml), trimethoprim-sulfamethoxazole (MIC90 1/19 micrograms/ml), and rifampin (MIC90 1 microgram/ml). Erythromycin (MIC90 greater than 8 micrograms/ml) and vancomycin (MIC90 greater than 16 micrograms/ml) demonstrated no activity. In vitro synergy (fractional inhibitory concentration index less than 0.5) was demonstrated with tetracycline plus rafampin in six of eight isolates tested.

Antimicrobial susceptibility of clinical isolates of Haemophilus influenzae to ampicillin-sulbactam.

A total of 1092 clinical isolates of Haemophilus influenzae (306 type b; 786 non-type-b), from five medical centers were obtained during 1987 and 1988. Disk diffusion antimicrobial susceptibilities were obtained for all isolates, and broth microdilution susceptibilities were obtained for 502 isolates. Beta-lactamase was produced by 34.3% of type-b and 22.1% of non-type-b isolates, with some geographic variations. Using disk diffusion antimicrobial susceptibility testing, all isolates were susceptible to ampicillin-sulbactam, ceftriaxone, cefuroxime, and rifampin; two isolates were resistant to chloramphenicol. Whether tested using a fixed ratio of ampicillin to sulbactam of 2:1 or a fixed concentration of sulbactam, the ampicillin-sulbactam combination demonstrated good activity against clinical isolates of H. influenzae. Only 8 of the 1092 isolates did not produce beta-lactamase but demonstrated MICs of greater than or equal to 2 micrograms/ml for ampicillin.

Ribosomal RNA gene (rrn) organization in enterococci.

A cloned 1.8-kb probe containing the 3' end of 16S ribosomal RNA and the 5' end of 23S ribosomal RNA from Enterococcus hirae was used to analyze various endonuclease digests of enterococci. In the ATCC strains tested we observed a remarkable conservation of the ApaI sites in the rrn operons, and a partial conservation of EcoRI sites. Using a number of other endonuclease digestions with the ApaI rrn probe, we estimate the number of rrn operons in enterococci to be between five and six.

Effect of capsulation on the resistance of Staphylococcus aureus to the bactericidal lipids produced in abscesses.

Staphylococcus aureus strains differ in their sensitivity to some of the bactericidal lipids produced by the host in staphylococcal abscesses. To evaluate whether the presence of a capsule might account for these differences, capsulate and non-capsulate S. aureus strains were compared for their sensitivity to staphylococcal abscess homogenates and the neutral lipid fraction derived from such material. Although the presence of a capsule appeared to reduce sensitivity, two non-capsulate mutants were only about three-to-four times more sensitive than their capsulate parent strains. Another strain, known for its resistance to these bactericidal lipids, was not capsulate. This suggests that mechanisms other than capsule formation must also determine sensitivity to the lipids.