Davise H. Larone

Clinical outcomes of patients with Klebsiella pneumoniae carbapenemase-producing K. pneumoniae after treatment with imipenem or meropenem

Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae may appear susceptible to imipenem or meropenem by routine susceptibility testing. We report a series of patients with infections caused by K. pneumoniae isolates, which yielded imipenem-susceptible results but were subsequently KPC-positive by polymerase chain reaction. When these infections were treated with imipenem or meropenem, frequent clinical and microbiologic failures were observed.

Relationship between semen cultures, leukospermia, and semen analysis in men undergoing fertility evaluation

OBJECTIVE To determine the correlation between semen cultures, semen Gram stains, leukospermia, and semen analysis parameters in asymptomatic men undergoing fertility evaluation. DESIGN Prospective clinical study. SETTINGS Infertility clinic at a university teaching hospital. PATIENT(S) Two hundred ninety-nine asymptomatic men undergoing fertility evaluation. INTERVENTION(S) After an antibacterial skin preparation, all men gave semen specimens for culture, Gram stains, semen analysis, and assessment for leukospermia. MAIN OUTCOME MEASURE(S) A comparison of the presence or absence of bacteriospermia, the specific bacterial isolate, the quantity of bacteriospermia, the presence or absence of leukospermia, and semen analysis parameters. RESULT(S) The presence or absence of bacteriospermia or leukospermia did not correlate with each other. Leukospermia did not correlate with semen parameters. Staphylococcus species was the most common isolate (25.4% of all patients) and did not correlate with semen parameters or leukospermia. Streptococcus viridans (15.4% of all patients) and Enterococcus faecalis (7.4% of all patients) were the next most common isolates and were associated with statistically significantly poorer semen quality. CONCLUSION(S) Leukospermia is a poor marker for either bacteriospermia or impaired semen quality. Staphylococcus species are commonly isolated but appear to be innocuous. Streptococcus viridans and Enterococcus faecalis are associated with poorer semen quality and may warrant treatment.

Multiplexed Identification of Blood-Borne Bacterial Pathogens by Use of a Novel 16S rRNA Gene PCR-Ligase Detection Reaction-Capillary Electrophoresis Assay

ABSTRACT We have developed a novel high-throughput PCR-ligase detection reaction-capillary electrophoresis (PCR-LDR-CE) assay for the multiplexed identification of 20 blood-borne pathogens (Staphylococcus epidermidis, Staphylococcus aureus, Bacillus cereus, Enterococcus faecalis, Enterococcus faecium, Listeria monocytogenes, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Escherichia coli, Klebsiella pneumoniae, Haemophilus influenzae, Pseudomonas aeruginosa, Acinetobacter baumannii, Neisseria meningitidis, Bacteroides fragilis, Bacillus anthracis, Yersinia pestis, Francisella tularensis, and Brucella abortus), the last four of which are biothreat agents. The method relies on the amplification of two regions within the bacterial 16S rRNA gene, using universal PCR primers and querying the identity of specific single-nucleotide polymorphisms within the amplified regions in a subsequent LDR. The ligation products vary in color and size and are separated by CE. Each organism generates a specific pattern of ligation products, which can be used to distinguish the pathogens using an automated software program we developed for that purpose. The assay has been verified on 315 clinical isolates and demonstrated a detection sensitivity of 98%. Additionally, 484 seeded blood cultures were tested, with a detection sensitivity of 97.7%. The ability to identify geographically variant strains of the organisms was determined by testing 132 isolates obtained from across the United States. In summary, the PCR-LDR-CE assay can successfully identify, in a multiplexed fashion, a panel of 20 blood-borne pathogens with high sensitivity and specificity.

Prospective, Multicenter Surveillance Study of Candida glabrata: Fluconazole and Itraconazole Susceptibility Profiles in Bloodstream, Invasive, and Colonizing Strains and Differences between Isolates from Three Urban Teaching Hospitals in New York City (Candida Susceptibility Trends Study, 1998 to 1999)

ABSTRACT Since the 1990s, the substantial increase in the rate of Candida glabrata infections has become a serious problem. As most C. glabrata infections arise from the hosts endogenous microflora, the present prospective, multicenter analysis included all clinical isolates associated with colonization and with systemic and hematogenous candidiasis. Among 347 C. glabrata isolates, the overall rates of resistance to fluconazole (MIC ≥ 64 μg/ml) and itraconazole (MIC ≥ 1 μg/ml) were 10.7 and 15.2%, respectively, although for half (n = 148) of the itraconazole-susceptible isolates the MICs (0.25 to 0.5 μg/ml) were in the susceptible—dependent upon dose range. Fluconazole resistance was more common among C. glabrata isolates obtained from centers caring for patients with cancer (MICs at which 90% of isolates are inhibited [MIC90s] = 32 μg/ml) or AIDS (MIC90s > 64 μg/ml) than among C. glabrata isolates from a community-based university medical center (MIC90s = 16 μg/ml) (P = 0.001). Thirty-three bloodstream isolates and those obtained from other body sites had similar in vitro susceptibility profiles. The fluconazole MIC90s (≤16 μg/ml) for C. glabrata yeast isolates from the gastrointestinal tract were lower than those (≥64 μg/ml) for C. glabrata isolates from respiratory and urinary tract samples (P = 0.01). A similar discrepancy for itraconazole was not significant (P > 0.5). We did not observe differences in fluconazole or itraconazole susceptibility profiles among C. glabrata isolates associated with either hematogenous dissemination or colonization. The significant discrepancy in antifungal susceptibility among C. glabrata organisms isolated from hospitals in the same geographic region emphasizes the significance of periodic susceptibility surveillance programs for individual institutions, especially those providing care to patients at risk.

Detection and Serotyping of Dengue Virus in Serum Samples by Multiplex Reverse Transcriptase PCR-Ligase Detection Reaction Assay

ABSTRACT The detection and successful typing of dengue virus (DENV) from patients with suspected dengue fever is important both for the diagnosis of the disease and for the implementation of epidemiologic control measures. A technique for the multiplex detection and typing of DENV serotypes 1 to 4 (DENV-1 to DENV-4) from clinical samples by PCR-ligase detection reaction (LDR) has been developed. A serotype-specific PCR amplifies the regions of genes C and E simultaneously. The two amplicons are targeted in a multiplex LDR, and the resultant fluorescently labeled ligation products are detected on a universal array. The assay was optimized using 38 DENV strains and was evaluated with 350 archived acute-phase serum samples. The sensitivity of the assay was 98.7%, and its specificity was 98.4%, relative to the results of real-time PCR. The detection threshold was 0.017 PFU for DENV-1, 0.004 PFU for DENV-2, 0.8 PFU for DENV-3, and 0.7 PFU for DENV-4. The assay is specific; it does not cross-react with the other flaviviruses tested (West Nile virus, St. Louis encephalitis virus, Japanese encephalitis virus, Kunjin virus, Murray Valley virus, Powassan virus, and yellow fever virus). All but 1 of 26 genotypic variants of DENV serotypes in a global DENV panel from different geographic regions were successfully identified. The PCR-LDR assay is a rapid, sensitive, specific, and high-throughput technique for the simultaneous detection of all four serotypes of DENV.

Development of Multiplex PCR-Ligase Detection Reaction Assay for Detection of West Nile Virus

ABSTRACT We have developed a novel multiplex reverse transcription-PCR ligase detection reaction (RT-PCR/LDR) assay for the detection of West Nile virus (WNV) in both clinical and mosquito pool samples. The method relies on the amplification of three different genomic regions, one in the coding sequence of nonstructural protein NS2a and two in nonstructural protein NS5, to minimize the risk of detection failure due to genetic variation. The sensitivity of the PCR is complemented by the high specificity of the LDR step, and the detection of the LDR products can be achieved with capillary electrophoresis (CE) or a universal DNA microarray. We evaluated the limit of detection by both one-step and two-step multiplex RT-PCR/LDR/CE approaches, which reached, respectively, 0.005 and 0.017 PFU. The assay demonstrated 99% sensitivity when mosquito pool samples were tested and 100% sensitivity with clinical samples when the one-step approach was used. The broad strain coverage was confirmed by testing 34 WNV isolates belonging to lineages 1 and 2, and the high specificity of the assay was determined by testing other flaviviruses, as well as negative mosquito pool and clinical samples. In summary, the multiplex RT-PCR/LDR assay could represent a valuable complement to WNV serological diagnosis, especially in early symptomatic patients. In addition, the multiplexing capacity of the technique, which can be coupled to universal DNA microarray detection, makes it an amenable tool to develop a more comprehensive assay for viral pathogens.

Klebsiella pneumoniae Isolate Producing at Least Eight Different β-Lactamases, Including AmpC and KPC β-Lactamases

Reports of the production of multiple β-lactamases in a single gram-negative pathogen are increasing (1, 2, 6). Isolates of Klebsiella species producing KPC-2, SHV extended-spectrum β-lactamases (ESBLs), and inhibitor-resistant TEM-30 β-lactamases have been reported as endemic in New York City (1). The present report identifies a Klebsiella pneumoniae isolate from New York City which produced up to 10 different β-lactamases, including a FOX-like plasmid-mediated AmpC, in addition to the previously reported KPC, SHV ESBL, and IRT β-lactamases (1). The K. pneumoniae isolate was obtained from the sputum of a patient with a mesothelioma. No carbapenem antibiotics were given to the patient prior to the isolation of the K. pneumoniae isolate. CLSI (formerly NCCLS) disk diffusion and broth microdilution assays (4) revealed that the K. pneumoniae isolate lacked susceptibility to levofloxacin (>16 μg/ml), amikacin (32 μg/ml), piperacillin-tazobactam (>128 μg/ml), ceftazidime (128 μg/ml), cefotaxime (64 μg/ml), aztreonam (>128 μg/ml), and cefoxitin (>64 μg/ml) but was susceptible by microbroth tests to tigecycline (0.5 μg/ml), minocycline (4 μg/ml), polymyxin B (1 μg/ml), cefepime, and imipenem. However, when broth microdilution tests were performed with an inoculum of 107 CFU/ml instead of 105 CFU/ml, the MICs of both imipenem and cefepime increased (4 μg/ml to 32 μg/ml and 8 μg/ml to >128 μg/ml, respectively). The MICs of tigecycline, minocycline, and polymyxin B were not affected when a higher inoculum was used. The presence of an ESBL was indicated by the CLSI ESBL confirmatory disk tests but not by the CLSI ESBL broth microdilution confirmatory tests (4). Isoelectric focusing (IEF) indicated that this isolate produced up to 10 different β-lactamases. Characterization of these enzymes was performed, as previously described, using IEF, inhibitor profiles, cefotaxime gel hydrolysis assays, and PCR (5-7, 8, 9). Using these techniques, 8 of 10 β-lactamases were identified. Three of these enzymes hydrolyzed cefotaxime. The pI and inhibitor profiles of these enzymes suggested the production of an SHV-12-like ESBL, a KPC-like carbapenem-hydrolyzing enzyme, a FOX-like AmpC, a PSE-1-like β-lactamase, and an OXA β-lactamase. In addition, TEM-1-like, TEM-30-like, and SHV-1-like enzymes were also identified (Table ​(Table1).1). PCR amplification using family-specific primers substantiated the presence of these genes within the K. pneumoniae isolate and identified the OXA β-lactamase as an OXA-9-like β-lactamase. Sequence data generated using the same primers that amplified the blaOXA product also suggested that the gene was blaOXA-9. TABLE 1. Identification of the β-lactamases produced by the K. pneumoniae isolate Accurate β-lactam susceptibility testing can be expected to become increasingly difficult over the next few years due to an increase in isolates producing multiple β-lactamases. The numbers and types of β-lactamases produced by the K. pneumoniae isolate from New York described in this report are a cause for concern, especially with respect to detecting ESBL, AmpC, and KPC-type enzymes. The CLSI microbroth ESBL confirmatory tests were unable to detect the presence of the SHV ESBL in this isolate, and these tests also suggested imipenem susceptibility, even though the isolate produced a KPC-type enzyme. As the number of pathogens producing multiple β-lactamases continues to rise, the difficulties in identifying the mechanisms responsible for β-lactam MICs will increase (1, 2, 6, 10). Clinical laboratories need the option of molecular testing in addition to phenotypic testing for the identification of resistance mechanisms that may be masked by the production of multiple enzymes. Additional testing options for these highly resistant pathogens may help avert future outbreaks like the ones reported for KPC-producing K. pneumoniae isolates in New York (1-3).

CHROMagar Candida as the sole primary medium for isolation of yeasts and as a source medium for the rapid-assimilation-of-trehalose test.

ABSTRACT The chromogenic medium BBL CHROMagar Candida (CAC) was evaluated as a sole primary medium for the isolation of yeasts from clinical specimens in which yeasts are the primary concern. Additionally, the reliability of the rapid-assimilation-of-trehalose (RAT) test in yielding correct results with isolates taken from CAC was assessed. A total of 270 throat, urine, and genital (TUG) specimens were streaked onto CAC, Sabouraud dextrose agar (SDA), inhibitory mold agar (IMA), and Mycosel (MYC). A total of 69 blood culture broths that were smear positive for yeast were streaked onto CAC and SDA. A 1-h RAT test (NCCLS M35-A) was performed simultaneously on isolates from CAC and SDA. A total of 112 TUG specimens yielded yeast colonies (CAC, 111 colonies; IMA, 105; SDA, 103; MYC, 91). The 69 blood culture yeasts grew on both CAC and SDA. Mixed cultures of yeasts were detected on 11 CAC plates but were unrecognized on other media. Colonies suspected of being C. glabrata on 32 CAC plates were all RAT test positive and confirmed to be C. glabrata; of 59 colonies with various characteristics of color and morphology on CAC, none were RAT positive, and all were conventionally identified as yeasts other than C. glabrata (sensitivity and specificity, 100%). The same isolates from SDA tested for RAT produced six false negatives and no false positives (sensitivity, 81%; specificity, 100%). The results show that CAC can be used as the sole primary medium for recovery of yeasts from clinical specimens. Additionally, isolates grown on CAC yield excellent results with the RAT test utilized in this study.

Medically Important Fungi: A Guide to Identification, Fifth Edition

Medically Important Fungi: A Guide to Identification, 5th Edition - Libros de Medicina - Microbiologia clinica - 89,30

Evaluation of MicroScan Autoscan for Identification of Pseudomonas aeruginosa Isolates from Cystic Fibrosis Patients

ABSTRACT Accurate identification of gram-negative bacilli from cystic fibrosis (CF) patients is essential. Only 57% (108 of 189) of nonmucoid strains and 40% (24 of 60) of mucoid strains were definitively identified as Pseudomonas aeruginosa with MicroScan Autoscan. Most common misidentifications were Pseudomonas fluorescens-Pseudomonas putida (i.e., the strain was either P. fluorescens or P. putida, but the system did not make the distinction and yielded the result P. fluorescens/putida) and Alcaligenes spp. Extending the incubation to 48 h improved identification, but 15% of isolates remained misidentified. The MicroScan Autoscan system cannot be recommended for the identification of P. aeruginosa isolates from CF patients.