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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.

The comparative in vitro potency of XRP2868, a new oral semisynthetic streptogramin antibiotic, was evaluated against gram-positive bacteria. XRP2868 inhibited all staphylococci at ≤1 μg/ml and all nonpneumococcal streptococci at ≤0.25 μg/ml and was fourfold more potent than quinupristin-dalfopristin against Staphylococcus aureus and Enterococcus faecium .

The activities of two investigational fluoroquinolones and three fluoroquinolones that are currently marketed were determined for 182 clinical isolates of Streptococcus pneumoniae . The collection included 57 pneumococcal isolates resistant to levofloxacin (MIC ≥ 8 μg/ml) recovered from patients in North America and Europe. All isolates were tested with clinafloxacin, gatifloxacin, gemifloxacin, levofloxacin, and trovafloxacin by the National Committee for Clinical Laboratory Standards broth microdilution and disk diffusion susceptibility test methods. Gemifloxacin demonstrated the greatest activity on a per gram basis, followed by clinafloxacin, trovafloxacin, gatifloxacin, and levofloxacin. Scatterplots of the MICs and disk diffusion zone sizes revealed a well-defined separation of levofloxacin-resistant and -susceptible strains when the isolates were tested against clinafloxacin and gatifloxacin. DNA sequence analyses of the quinolone resistance-determining regions of gyrA , gyrB , parC , and parE from 21 of the levofloxacin-resistant strains identified eight different patterns of amino acid changes. Mutations among the four loci had the least effect on the MICs of gemifloxacin and clinafloxacin, while the MICs of gatifloxacin and trovafloxacin increased by up to six doubling dilutions. These data indicate that the newer fluoroquinolones have greater activities than levofloxacin against pneumococci with mutations in the DNA gyrase or topoisomerase IV genes. Depending upon pharmacokinetics and safety, the greater potency of these agents could provide improved clinical efficacy against levofloxacin-resistant pneumococcal strains.

Klebsiella pneumoniae K6 (ATCC 700603), a clinical isolate, is resistant to ceftazidime and other oxyimino-β-lactams. A consistent reduction in the MICs of oxyimino-β-lactams by at least 3 twofold dilutions in the presence of clavulanic acid confirmed the utility of K. pneumoniae K6 as a quality control strain for extended-spectrum β-lactamase (ESBL) detection. Isoelectric-focusing analysis of crude lysates of K6 demonstrated a single β-lactamase with a pI of 7.8 and a substrate profile showing preferential hydrolysis of cefotaxime compared to ceftazidime. PCR analysis of total bacterial DNA from K6 identified the presence of a bla SHV gene. K6 contained two large plasmids with molecular sizes of approximately 160 and 80 kb. Hybridization of plasmid DNA with a bla SHV -specific probe indicated that a bla SHV gene was encoded on the 80-kb plasmid, which was shown to transfer resistance to ceftazidime in conjugal mating experiments with Escherichia coli HB101. DNA sequencing of this bla SHV -related gene revealed that it differs from bla SHV-1 at nine nucleotides, five of which resulted in amino acid substitutions: Ile to Phe at position 8, Arg to Ser at position 43, Gly to Ala at position 238, and Glu to Lys at position 240. In addition to the production of this novel ESBL, designated SHV-18, analysis of the outer membrane proteins of K6 revealed the loss of the OmpK35 and OmpK37 porins.

The presence of Enterococcus -associated vancomycin resistance genes vanA , vanB , vanD , vanE , and vanG in rectal swabs was investigated in two hospitals using PCR. All vanA genes detected were associated with vancomycin-resistant enterococci (VRE), whereas VRE-associated vanB genes were detected in only one hospital (4.7%). However, in both hospitals, high prevalences of vanB (6.2 and 2.3%), vanD (43.8 and 26.7%), and vanG (10.5 and 6.9%) genes not associated with enterococci were found.

The optimal diagnostic approach to pneumonia provides a rapid microbiologic identification of pulmonary pathogens by the least invasive means. The technique of sputum induction has been useful in the evaluation of patients with Pneumocystis carinii or mycobacterial pneumonia. It is not known whether induced sputum samples are preferable for the detection of pathogens other than P. carinii or mycobacteria. Microbiologic yields were evaluated from identically processed induced and conventional sputum samples collected from 509 consecutive patients. No statistically significant differences were found between the microbiologic yields of induced and spontaneous sputum samples. Bacterial pathogens were isolated in 19.6% of induced and 23.5% of routine specimens. Mycobacteria were cultured from 13.1% of routine and 9.4% of induced specimens. Non-Candida albicans fungi grew from 24% of routine and 20% of induced specimens. The process of sputum induction with aerosolized hypertonic saline did not alter either the purulence or the bacterial quantitation of Gram-stained sputum specimens. Sputum induction has been useful for the cytologic diagnosis of malignancy, for the diagnosis of pneumocystosis and tuberculosis, and in patients unable to spontaneously produce sputum samples. The use of induced sputum samples for the diagnosis of other infections may not be necessary when routine sputum specimens are available.

Members of the Streptococcus bovis group are important causes of endocarditis. However, factors associated with their pathogenicity, such as adhesins, remain uncharacterized. We recently demonstrated that endocarditis-derived Streptococcus gallolyticus subsp. gallolyticus isolates frequently adhere to extracellular matrix (ECM) proteins. Here, we generated a draft genome sequence of an ECM protein-adherent S. gallolyticus subsp. gallolyticus strain and found, by genome-wide analyses, 11 predicted LPXTG-type cell wall-anchored proteins with characteristics of MSCRAMMs, including a modular architecture of domains predicted to adopt immunoglobulin (Ig)-like folding. A recombinant segment of one of these, Acb, showed high-affinity binding to immobilized collagen, and cell surface expression of Acb correlated with the presence of acb and collagen adherence of isolates. Three of the 11 proteins have similarities to major pilus subunits and are organized in separate clusters, each including a second Ig-fold-containing MSCRAMM and a class C sortase, suggesting that the sequenced strain encodes three distinct types of pili. Reverse transcription-PCR demonstrated that all three genes of one cluster, acb - sbs7 - srtC1 , are cotranscribed, consistent with pilus operons of other gram-positive bacteria. Further analysis detected expression of all 11 genes in cells grown to mid to late exponential growth phases. Wide distribution of 9 of the 11 genes was observed among S. gallolyticus subsp. gallolyticus isolates with fewer genes present in other S. bovis group species/subspecies. The high prevalence of genes encoding putative MSCRAMMs and pili, including a collagen-binding MSCRAMM, among S. gallolyticus subsp. gallolyticus isolates may play an important role in the predominance of this subspecies in S. bovis endocarditis.

Members of the Streptococcus bovis group are frequent colonizers of the intestinal tract, which can also cause endocarditis. However, their ability to adhere to and colonize host tissues and the factors associated with pathogenicity are largely unknown. Here, we assessed 17 endocarditis-derived human isolates [identified here as 15 Streptococcus gallolyticus ssp. gallolyticus (S. bovis biotype I), one S. gallolyticus ssp. pasteurianus (biotype II/2) and one Streptococcus infantarius ssp. coli (biotype II/1)] for their in vitro adherence to components of the extracellular matrix (ECM). Adherence to collagen type I was found to be the most common phenotype exhibited by 76% of isolates, followed by collagen type IV (53%), fibrinogen (47%), collagen type V (35%) and fibronectin (35%). Pulsed-field gel electrophoresis analyses showed that >50% of endocarditis-derived S. gallolyticus ssp. gallolyticus isolates are genetically diverse, although two clusters of two and four isolates were observed. The diversity of strains and differences observed in adherence characteristics to distinct host ECM proteins suggest that isolates of S. gallolyticus ssp. gallolyticus produce different surface components, similar to other gram-positive pathogens, to colonize the host and cause infection.

We reevaluated Enterobacteriaceae disk diffusion breakpoints for the tetracyclines published in the Clinical and Laboratory Standards Institute (CLSI) document M100-S16, which were (susceptible/resistant) ≥19 mm/≤14 mm for tetracycline, ≥16 mm/≤12 mm for doxycycline, and ≥19 mm/≤14 mm for minocycline. A collection of 504 recent clinical isolates of Enterobacteriaceae were tested against these tetracycline compounds by disk diffusion and broth microdilution methods according to CLSI guidelines. Regression line and scattergram plot analyses determined intermethod accuracy for current disk diffusion breakpoints and showed excellent r values of 0.91 to 0.95. However, error rates (minor/major [false-resistant]/very major [false-susceptible]) were 14.9/0.8/0.0% for tetracycline, 11.5/0.4/0.0% for doxycycline, and 30.6/0.7/0.0% for minocycline and only 4.4/0.0/0.0% for tetracycline, 5.6/0.0/0.2% for doxycycline, and 8.3/0.0/0.3% for minocycline when proposed breakpoints were modified to (susceptible/resistant) ≥15 mm/≤11 mm for tetracycline, ≥14 mm/≤10 mm for doxycycline, and ≥16 mm/≤12 mm for minocycline. Listed modifications were recently approved by the CLSI (M100-S17).

Streptococcus bovis causes 24% of all streptococcal infective endocarditis cases. There are many reports linking both S. bovis bacteremia and endocarditis with various forms of gastrointestinal disease (primarily colonic cancers). S. bovis is divided into two biotypes: I and II. The biotype I strain is much more frequently isolated from patients with endocarditis, gastrointestinal disease, or both. We describe here the isolation of biotype I-specific DNA sequences and the development of a PCR test which can identify S. bovis biotype I strains among S. bovis clinical isolates.

Resistance to fluoroquinolone (FQ) antibiotics in Streptococcus pneumoniae has been attributed primarily to specific mutations in the genes for DNA gyrase ( gyrA and gyrB ) and topoisomerase IV ( parC and parE ). Resistance to some FQs can result from a single mutation in one or more of the genes encoding these essential enzymes. A group of 160 clinical isolates of pneumococci was examined in this study, including 36 ofloxacin-resistant isolates (MICs, ≥8 μg/ml) recovered from patients in North America, France, and Belgium. The susceptibilities of all isolates to clinafloxacin, grepafloxacin, levofloxacin, sparfloxacin, and trovafloxacin were examined by the National Committee for Clinical Laboratory Standards reference broth microdilution and disk diffusion susceptibility testing methods. Among the ofloxacin-resistant strains, 32 of 36 were also categorized as resistant to levofloxacin, 35 were resistant to sparfloxacin, 29 were resistant to grepafloxacin, and 19 were resistant to trovafloxacin. In vitro susceptibility to clinafloxacin appeared to be least affected by resistance to the other FQs. Eight isolates with high- and low-level resistance to the newer FQs were selected for DNA sequence analysis of the quinolone resistance-determining regions (QRDRs) of gyrA , gyrB , parC , and parE . The DNA and the inferred amino acid sequences of the resistant strains were compared with the analogous sequences of reference strain S. pneumoniae ATCC 49619 and FQ-susceptible laboratory strain R6. Reduced susceptibilities to grepafloxacin and sparfloxacin (MICs, 1 to 2 μg/ml) and trovafloxacin (MICs, 0.5 to 1 μg/ml) were associated with either a mutation in parC that led to a single amino acid substitution (Ser-79 to Phe or Tyr) or double mutations that involved the genes for both GyrA (Ser-81 to Phe) and ParE (Asp-435 to Asn). High-level resistance to all of the compounds except clinafloxacin was associated with two or more amino acid substitutions involving both GyrA (Ser-81 to Phe) and ParC (Ser-79 to Phe or Ser-80 to Pro and Asp-83 to Tyr). No mutations were observed in the gyrB sequences of resistant strains. These data indicate that mutations in pneumococcal gyrA , parC , and parE genes all contribute to decreased susceptibility to the newer FQs, and genetic analysis of the QRDR of a single gene, either gyrA or parC , is not predictive of pneumococcal resistance to these agents.

Estradiol USP was extemporaneously compounded for intravenous administration. Eight postmenopausal women were randomized to receive one of four estradiol dosages. Serum estradiol concentrations were determined at frequent intervals after single bolus dosing. The concentration-time profile was stripped and fit, and pharmacokinetic values were generated. Approximate dosage proportionality was seen with area under the curve, the terminal half-life was 27.45+/-5.65 minutes, and volume of distribution was very low (0.082+/-0.015 L/kg). Estradiol was well tolerated by all study participants.

Intracoronary testosterone injections have recently been shown to possess coronary vasodilating effects. The same may be true for intravenous testosterone, but the pharmacokinetic and hemodynamic aspects need exploration before pharmacologic studies can begin. This trial determined the pharmacokinetic and hemodynamic properties of 300 microg of testosterone given intravenously. Degree of testosterone aromatization to 17-beta estradiol after exogenous administration and overall patient tolerability also were evaluated. Eleven elderly men with coronary artery disease participated in the study and were given 300 microg of testosterone intravenously over 10 minutes. Serum blood concentrations of testosterone and 17-beta estradiol were measured at baseline and then periodically. Testosterone serum concentrations were stripped and fit to a two-compartment model for all patients. The volume of distribution (Vdarea) was 80.36 +/- 24.51 L, and the elimination half-life was 55.93 +/- 23.06 minutes. No hemodynamic differences or side effects were noted. The serum concentrations of 17-beta estradiol were significantly increased from baseline beginning 5 minutes after infusion to the end of the study (180 minutes after infusion).

Factors contributing to the recent upswing in tuberculosis (TB) cases in the United States include the shifting of management of TB cases from specialized hospital units to lesser-equipped outpatient settings; reduced medical school teaching of TB-related issues; large-scale immigration from regions endemic for TB; and the homelessness and AIDS cases that have created a pocket of disease in high-risk populations. At least 33 percent of current cases can be attributed to new rather than reactivated infection, and many of these are caused by the ever-increasing number of drug-resistant strains. Multidrug-resistant TB has a higher overall fatality rate and disproportionately affects immunocompromised and HIV-infected individuals. Clearly, expedient diagnosis is important in controlling the spread of tuberculosis. Sputum samples, evaluated first by direct microscopic evaluation (smear), are visualized with either the easily detected acid-fast fluorochrome dye auramine O, or the more specific Ziehl-Neelsen stain. Specimens are cultured on either solid media (Lowenstein-Jensen slant), or are grown in a liquid medium, such as the BACTEC automated radiometric system. Next, biochemical or nucleic acid probe testing is used to identify various strains. Isolates are tested for resistance to commonly used antituberculosis drugs, often by using the new method of susceptibility testing in liquid broth rather than the traditional agar dilution method. Clinical laboratories await FDA approval of two new methods employing hybridization with rRNA genes or polymerase chain reaction (PCR) that will further speed up diagnosis of TB.

Plasmids encoding extended-spectrum beta-lactamases of the TEM, SHV, and AmpC families were introduced into common Escherichia coli and Klebsiella pneumoniae hosts to create a homogeneous panel for evaluating the abilities of five test systems to detect resistance to eight beta-lactam antibiotics. Although MICs, as determined by agar dilution or E test strips, were increased and disk diffusion zone diameters were diminished, breakpoints for resistance were often not reached, and neither approach was sensitive in detecting resistance to oxyimino-beta-lactams. The MicroScan 18-h microdilution or Vitek rapid automated procedures were similarly insensitive. Ceftazidime was the best single test antibiotic for detecting extended-spectrum beta-lactamase production. beta-Lactamases TEM-7 and TEM-12 were particularly difficult to detect. Because of such difficulties, the prevalence of extended-spectrum beta-lactamases is likely to be greater than is currently appreciated.

We evaluated the in vitro activity of piperacillin alone or in combination with the beta-lactamase inhibitor tazobactam against clinical isolates of Legionella species. At an inoculum of approximately 10(4) CFU, tazobactam, piperacillin, and the 8:1 combination had equivalent activities against Legionella spp. At an approximately 10-fold higher inoculum, the following results were obtained, expressed as MICs for 50 and 90% of strains tested (MIC range): piperacillin, 4 and 16 (0.25 to 32) micrograms/ml; tazobactam, 0.5 and 1 (0.125 to 2) micrograms/ml; and piperacillin-tazobactam (expressed in terms of MIC of piperacillin) 0.5 and 1 (0.03 to 2) micrograms/ml. Tazobactam alone and the combination with piperacillin were more active than piperacillin alone at the higher inoculum.

Nine hundred forty-five stool specimens from patients suspected of having Clostridium difficile disease were examined using a cell culture cytotoxicity assay (CTA), two enzyme immunoassay (EIA) kits (Cytoclone for toxins A and B; VIDAS for toxin A) and a latex agglutination assay (CDT). One hundred nineteen specimens had positive titers (or = 90) in the CTA; clinical review of 16 discordant samples and 49 controls supported the significance of 90 as the positive cut-off titer. The performance of the two EIAs and the latex assay was assessed relative to CTA titers of the samples. Sensitivity wasor = 50% for all three assays for the 24 specimens with CTA titers of 90, but it reached 97-100% for the two EIAs and 84% for the latex assay at titers ofor = 2,250. The Cytoclone EIA exhibited higher sensitivity at the lower positive titers. Overall, specificity of the methods ranged from 96.7% (CDT latex assay) to 99.1% (Cytoclone EIA).

We evaluated a rapid immunoperoxidase technique for the detection of cytomegalovirus (CMV) antigenemia in peripheral blood neutrophils of 56 transplant recipients (117 specimens) and 36 persons with AIDS (59 specimens). Antigenemia was 92% sensitive and 98% specific for the detection of clinical CMV infection in transplant recipients and 100% sensitive and 86% specific in persons with AIDS. Overall, CMV antigenemia was a more rapid and sensitive method for the detection of clinical CMV infection than either shell vial culture or conventional tube culture of blood.

The number of patients who are at an increased risk of life-threatening infection has expanded greatly during the past decade. This expansion is because of two events, one terrible in its consequences, the other very positive. The first is the unprecedented epidemic of opportunistic infection that follows infection with the human immunodeficiency virus or the acquired immunodeficiency syndrome. The second is a direct consequence of the successes of the transplant surgeon, the cancer chemotherapist, the hematologist, and the rheumatologist. The authors review the current issues and trends in understanding and diagnosing infectious complications in the immunocompromised host.

A retrospective study was performed to determine the clinical incidence and causes of infection related to the use of frozen musculoskeletal allografts. The results of this study of 324 grafts, prepared and supplied by our hospital bone bank, showed that the patients in whom femoral head grafts and other small bone and soft-tissue allografts were used had a negligible clinical incidence of infection. The incidence of infection related to the use of large allografts, such as osteoarticular or diaphyseal grafts, was approximately 5 per cent in patients who had treatment for a bone tumor and 4 per cent in those who had revision of a hip arthroplasty. These rates of infection were not substantially different from those that have been reported in similar series in which large allografts or sterilized prosthetic devices were used. The causes of the infections were difficult to determine, but contamination of the allograft was probably not a factor in most patients.

The fecal lactoferrin assay was more sensitive (75%) than methylene blue microscopy (40%) for the detection of leukocytes in Clostridium difficile toxin-positive fecal samples. Although limited sensitivity and specificity precludes its use as a laboratory screening test, it may be a more useful initial test in an algorithm for clinically suspected C. difficile-associated disease.

The susceptibility of 22 clinical isolates of Campylobacter pylori to eight antimicrobial agents was studied under varying pH conditions. Macrolides (erythromycin, dirythromycin), clindamycin and to a lesser extent quinolones lost efficacy at lowered pH. The activity of ampicillin and metronidazole remained relatively stable throughout the pH range tested. The effect of pH an antimicrobial efficacy may warrant consideration when selecting an antibiotic to clear Campylobacter pylori in vivo, since the pH of the gastric submucous environment is not known for certain.