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To rapidly identify Mycobacterium and Nocardia spp. without costly probes, we had implemented capillary electrophoresis (CE) in polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analysis to analyze their 65-kDa heat shock protein (hsp65) gene. The PCR-RFLP analysis with CE (PRACE) involved only one restriction enzyme, HaeIII, and a single electrophoretic separation less than 10 min. Full-range (10-200 bp) RFLP patterns of 12 less common Mycobacterium and 7 Nocardia spp. were investigated. A good agreement was observed between the sizes of restriction fragments resolved by CE and the real sizes deduced from sequence analysis. Including hsp65 gene patterns of 12 Mycobacterium spp. published earlier, differentiation was distinct among 24 Mycobacterium and 7 Nocardia spp. Some closely related species exhibiting similar biochemical characteristics could be well discriminated by an extra HaeIII digestion site. Thus, PRACE offers a nonprobe alternative for rapid identification of various cultured Mycobacterium and Nocardia to the species level.

The following case illustrates an ileal perforation and reactive hemophagocytic syndrome (RHS) resulting from disseminated histoplasmosis in a patient with Human Immunodeficiency Virus (HIV) from Puerto Rico. Although the diagnosis was established by histopathologic findings and a positive bone marrow culture, Histoplasma capsulatum-specific real-time Polymerase Chain Reaction (PCR) allowed to confirm the diagnosis from formalin-fixed, paraffin-embedded tissue. Interestingly, the Histoplasma antigens in both serum and urine samples were falsely negative. Amphotericin B lipid complex (Abelcet), followed by oral itraconazole, led to a successful response and resolution of symptoms. A short review of the clinical signs and symptoms, diagnostic tests, and therapeutic options for disseminated histoplasmosis is done, with emphasis on the role of Histoplasma-specific real-time PCR as a molecular diagnostic tool and the efficacy of treatment with one of the lipid formulations of amphotericin B.

A fully automated commercial antifungal susceptibility test system (VITEK 2 yeast susceptibility test; bioMerieux, Inc., Hazelwood, Mo.) was compared in three different laboratories with Clinical and Laboratory Standards Institute (CLSI) reference broth microdilution (BMD) method by testing two quality control strains and a total of 426 isolates of Candida spp. (103 to 135 clinical isolates in each laboratory plus 80 challenge isolates in one laboratory) against fluconazole. Reference BMD MIC endpoints were established after 24 and 48 h of incubation. VITEK 2 endpoints were determined spectrophotometrically after 10 to 26 h of incubation (mean, 13 h). Excellent essential agreement (within two dilutions) between the VITEK 2 and the 24- and 48-h BMD MICs was observed. The overall agreement values were 97.9 and 93.7%, respectively. Both intra- and interlaboratory agreement was 100%. The overall categorical agreement between VITEK 2 and BMD was 97.2% at the 24-h BMD time point and 88.3% at the 48-h BMD time point. Decreased categorical agreement at 48 h was attributed to trailing growth observed with Candida glabrata. The VITEK 2 system reliably detected fluconazole resistance among Candida spp. and demonstrated excellent quantitative and qualitative agreement with the reference BMD method.

In order to assess the speed and accuracy of a real-time PCR assay targeting the lukS-PV gene of Panton-Valentine leukocidin (PVL)-positive Staphylococcus aureus, 700 S. aureus strains were tested and the results were compared to those achieved with block cycler PCR. Cross-reactivity was tested with 166 other bacterial species. Using this homogeneous real-time PCR assay format, the presence or absence of genetic information for PVL, which is also found in community-associated methicillin-resistant S. aureus, was correctly identified from pure culture and directly in various types of clinical specimens.

We examined 49 Legionella species, 26 L. pneumophila and 23 non-pneumophila Legionella spp., using partial 16S rRNA gene sequencing. This approach accurately identified all the L. pneumophila isolates, characterized all non-pneumophila Legionella isolates as such within this genus, and classified most (20/23; 87%) of the non-pneumophila Legionella isolates to the species level.

Purpose: To describe a new technique, pyrosequencing, which allows for the rapid identification of Mycobacterium and Nocardia species., Design: Interventional case report., Methods: The medical records of a patient presenting with an infectious keratitis were reviewed., Results: A case of Nocardia abscessus/arthrititis/asiatica keratitis was diagnosed in a young individual with the aid of pyrosequencing technology. Based on presumed antibiotic sensitivities, therapy with topical trimethoprim-sulfamethoxazole eyedrops was initiated, and the infection was cleared rapidly with minimal residual scarring., Conclusions: Pyrosequencing may be a useful tool in aiding the rapid diagnosis and treatment of ocular infections caused by slow-growing pathogens.

We report a case of a culture-negative osteomyelitis in which our newly developed real-time polymerase chain reaction (PCR) could differentiate Staphylococcus aureus from Staphylococcus epidermidis. This is the first report that described the application of this novel assay to an orthopedics clinical sample. This assay may be useful for other clinical culture-negative cases in a combination with a broad-spectrum assay as a rapid microorganism identification method.

Recently published clinical trial data have produced compelling evidence for increased survival when Herceptin is administered to patients whose tumors are HER2 amplified. Therefore, the accuracy of HER2 status is essential to determine which patients should or should not receive Herceptin. Although HER2 results obtained by FISH and IHC are often in agreement, there is a persistent group of cases in which results are discordant, particularly among tumors with intermediate results. A multivariable analysis was undertaken to determine relative significance of various clinical and pathologic findings for patients diagnosed with infiltrating ductal carcinoma, and a data model was produced that predicts which patients are most likely to have HER2 amplified tumors. Correlates of HER2 amplification were higher Scarff-Bloom-Richardson grade, younger age at diagnosis, and a comedo ductal carcinoma in situ component.

Purpose: To determine the spectrum of organisms causing acute bacterial conjunctivitis in hospitalized children at a tertiary care referral center., Design: Retrospective chart review., Methods: Charts of hospitalized children with positive conjunctival cultures were reviewed, and patients with clinical description of conjunctivitis were studied., Results: One hundred and seven isolates from 59 patients were included in the study. The most common organisms cultured were coagulase-negative Staphylococcus (59.3% of patients), viridans Streptococcus (47.5%), and Staphylococcus aureus (20.3%). The type of organisms differed based on age, with S. aureus and Haemophilus influenzae being more common in nonneonates. Gram-negative bacilli and methicillin-resistant Staphylococcus species were more common in patients hospitalized longer than two days., Conclusions: The distribution of bacterial organisms causing acute bacterial conjunctivitis in our hospitalized children differs from that of previous reports in the outpatient setting. Conjunctival swabbing for culture and sensitivities before instituting empiric broad-spectrum antibiotic therapy may be useful.

We have developed a combined real-time PCR and pyrosequencing assay that successfully differentiated the vast majority of gram-positive and gram-negative bacteria when bacterial isolates were tested. The purpose of this study was to evaluate this assay on clinical specimens obtained from orthopedic surgeries, and to prospectively compare the results of "molecular Gram stain" with culture and conventional direct Gram stain. Forty-five surgical specimens were obtained from patients who underwent orthopedic surgery procedures. The DNA was extracted and a set of broad-range PCR primers that targeted a part of the 16S rDNA gene was used for pan-bacterial PCR. The amplicons were submitted for pyrosequencing and the resulting molecular Gram stain characteristics were recorded. Culture and direct Gram staining were performed using standard methods for all cases. Surgical specimens were reviewed histologically for all cases that had a discrepancy between culture and molecular results. There was an 86.7% (39/45) agreement between the traditional and molecular methods. In 12/14 (85.7%) culture-proven cases of bacterial infection, molecular Gram stain characteristics were in agreement with the culture results, while the conventional Gram stain result was in agreement only for five cases (35.7%). In the 31 culture negative cases, 27 cases were also PCR negative, whereas 4 were PCR positive. Three of these were characterized as gram negative and one as gram positive by this molecular method. Molecular determination of the Gram stain characteristics of bacteria that cause orthopedic infections may be achieved, in most instances, by this method. Further studies are necessary to understand the clinical importance of PCR-positive/culture-negative results.

The prognostic and therapeutic implications of HER-2 gene amplification and estrogen and progesterone receptor status in breast cancer are well described. To address the relative paucity of information concerning HER-2 amplification for tubular carcinomas, we assessed the frequency of gene amplification in 55 tubular carcinomas of the breast from 54 patients, 5 of which had axillary node metastases. The HER-2 gene copy number was assessed by fluorescence in situ hybridization for the majority of tumors analyzed, whereas estrogen and progesterone receptor status was achieved by immunohistochemical analysis. HER-2 gene amplification was not observed in any of the tumors examined, and most were estrogen receptor-positive. This HER-2 gene amplification frequency was significantly lower than the frequency of gene amplification previously reported for all invasive ductal carcinoma of no special type (P < .01). HER-2 gene amplification likely occurs infrequently, or not at all, in tubular carcinomas of the breast, whereas most express estrogen receptors.

Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease of the central nervous system caused by the JC polyoma virus. Electron microscopy and immunohistochemistry are the traditional methods of confirming the presence of the virus in brain biopsies from these patients. We studied the brain biopsies from 7 patients with PML and 6 patients without PML with chromogenic in situ hybridization (CISH) for the JC polyoma virus using a commercially available probe. The biopsies from the patients with the PML cases were proven to contain the JC polyoma virus by traditional and molecular methods. The CISH findings were compared with the known state of infection. All (7/7) of the biopsies from patients with PML were positive for the presence of polyoma virus by CISH, whereas the biopsies from patients without PML were uniformly negative. CISH seems to be a useful tool for the detection of the JC virus in brain biopsies from patients with PML, and is more accessible because a commercial probe is available.

Background: Indiscriminate antibiotic use may lead to development of antibiotic resistance. Preoperative mupirocin treatment decreases Staphylococcus aureus carriage and may reduce subsequent surgical site infection, but is unlikely to benefit noncarriers. This study was undertaken to evaluate whether avoiding mupirocin in noncarriers places them at increased risk for subsequent postoperative infection., Methods: We conducted a retrospective cohort study examining incidence of postoperative infection in patients undergoing cardiac surgery at the Cleveland Clinic after introduction of a protocol of polymerase chain reaction screening for nasal S aureus carriage, and avoiding mupirocin treatment of noncarriers., Results: Between August 1, 2002, and May 31, 2004, 6,334 patients were screened for nasal carriage of S aureus before undergoing cardiac surgery. There was no significant difference in infection rates between carriers and noncarriers when examining the incidence of all infections (5.6% and 5.0%; relative risk [RR] 1.11 [95% confidence interval (CI): 0.86 to 1.43]), infections caused specifically by S aureus (1.04% and 0.80%; RR 1.30 [95% CI: 0.71 to 2.39]), any surgical site infection (3.1% and 3.2%; RR 0.97 [95% CI: 0.69 to 1.36]), S aureus surgical site infection (0.82% and 0.58%; RR 1.41 [95% CI: 0.71 to 2.82]), any bloodstream infection (3.1% and 2.5%; RR 1.21 [95% CI: 0.86 to 1.71]), and S aureus bloodstream infection (0.37% and 0.48%; RR 0.77 [95% CI: 0.30 to 2.03]). Mupirocin use declined substantially after introduction of the protocol., Conclusions: A strategy of targeting perioperative mupirocin treatment to carriers leads to significant reduction in mupirocin use without increasing early postoperative infectious complications in noncarriers.

Objective: To describe the investigation and interventions necessary to contain an outbreak of methicillin-resistant Staphylococcus aureus (MRSA) colonization and infection in a neonatal intensive care unit (NICU)., Design: Retrospective case finding that involved prospective performance of surveillance cultures for detection of MRSA and molecular typing of MRSA by repetitive-sequence polymerase chain reaction (rep-PCR)., Setting: Level III NICU in a tertiary care center., Participants: Three neonates in a NICU were identified with MRSA bloodstream infection on April 16, 2004. A point prevalence survey identified 6 additional colonized neonates (attack rate, 75% [9 of 12 neonates]). The outbreak strain was phenotypically unusual., Interventions: Cohorting and mupirocin therapy were initiated for neonates who had acquired MRSA during the outbreak. Contact precautions were introduced in the NICU, and healthcare workers (HCWs) were retrained in cleaning and disinfection procedures and hand hygiene. Noncolonized neonates and newly admitted patients had surveillance cultures performed 3 times per week., Results: Two new colonized neonates were identified 1 month later. HCW X, who had worked in the NICU since June 2003, was identified as having chronic otitis. MRSA was isolated from cultures of swab specimens from HCW X's ear canal and nares. HCW X was epidemiologically linked to the outbreak. Molecular typing (by rep-PCR) confirmed that the isolates from HCW X and from the neonates were more than 90% similar. Retrospective review of NICU isolates revealed that the outbreak strain was initially cultured from a neonate 2 months after HCW X began working on the unit. The epidemic strain was eradicated after removing HCW X from patient care in the NICU., Conclusion: An outbreak of MRSA colonization and infection in a NICU was epidemiologically linked to a HCW with chronic otitis externa and nasal colonization with MRSA. Eradication was not achieved until removal of HCW X from the NICU. Routine surveillance for MRSA may have allowed earlier recognition of the outbreak and is now standard practice in our NICU.

Background: The isolation of a pathogen is vital in the diagnosis and treatment of a device infection. A swab culture, despite poor sensitivity, is the most common method used in specimen collection., Objective: To determine the relative value of swab and tissue specimen cultures in patients with implantable cardiac pacemakers and defibrillators., Design: Prospective patient cohort study., Setting: A 1,000-bed tertiary referral center in Cleveland, Ohio., Patients: Consecutive patients with implantable cardiac pacemaker or defibrillator presenting for lead extraction from October 1, 2000 to March 31, 2001., Methods: Tissue and swab cultures were prospectively collected during pacemaker and implantable defibrillator surgeries that required lead extraction. Clinical manifestations, microbiology, and echocardiographic data were recorded in patients with and without a clinical diagnosis of device system infection., Results: Seventy-one patients with implantable pacemaker (n = 49, 69%), implantable defibrillator (n = 18, 25%), or both devices (n = 4, 6%) requiring lead extraction had pocket swab and tissue cultures for analysis. Infection was evident clinically in 35 (49%) of the patients and absent in the remainder. The most common bacteria isolated were coagulase-negative Staphylococcus (37%) and Staphylococcus aureus (10%). Patients with clinical infection had positive cultures more frequently (P = 0.002) by pocket tissue culture (n = 24, 69%) than by swab culture (n = 11, 31%). However, patients without clinical infections had positive cultures at similar rates by pocket tissue culture (n = 10, 28%) and by swab culture (n = 8, 22%; P = 0.48). Patients without clinical infection were not treated with other than perioperative antibiotics, and did not develop clinical infections., Conclusion: Pocket tissue cultures are more effective than pocket swab cultures for the isolation and identification of the infectious pathogens in cardiac device infections. Positive cultures by pocket swab or tissue cultures in the absence of clinical signs and symptoms of infection does not imply infection or the need for specific therapy.

We evaluated the Roche LightCycler Staphylococcus M(GRADE) kits to differentiate between Staphylococcus aureus and coagulase-negative staphylococci in blood cultures growing clusters of gram-positive cocci. Testing 100 bottles (36 containing S. aureus), the assay was 100% sensitive and 98.44% specific for S. aureus and 100% sensitive and specific for coagulase-negative staphylococci.

West Nile virus (WNV) infections are potentially life threatening in immunocompromised hosts. Currently, the best diagnostic test is serology. Reverse-transcriptase polymerase chain reaction (RT-PCR) testing has a role, but, because WNV is a cell-associated neurotropic virus, RT-PCR results are frequently negative even in cases of active infection. We present a case in which serology results were persistently negative because the patient was immunocompromised following lymphoma treatment. The role of humoral immunity in resolution of WNV is also discussed.

A nucleic acid extraction system that can handle small numbers of specimens with a short test turnaround time and short hands-on time is desirable for emergent testing. We performed a comparative validation on three systems: the MagNA Pure compact system (Compact), the NucliSens miniMAG extraction instrument (miniMAG), and the BioRobot EZ1 system (EZ1). A total of 75 urine specimens submitted for polyomavirus BK virus detection were used. The human beta-actin gene was detected on 75 (100%), 75 (100%), and 72 (96%) nucleic acid extracts prepared by the miniMAG, EZ1, and Compact, respectively. The miniMAG produced the highest quantity of nucleic acids and the best precision among the three systems. The agreement rate was 100% for BKV detection on nucleic acid extracts prepared by the three extraction systems. When a full panel of specimens was run, the hands-on time and test turnaround time were 105.7 and 121.1 min for miniMAG, 6.1 and 22.6 min for EZ1, and 7.4 and 33.7 min for Compact, respectively. The EZ1 and Compact systems processed automatic nucleic acid extraction properly, providing a good solution to the need for sporadic but emergent specimen detection. The miniMAG yielded the highest quantity of nucleic acids, suggesting that this system would be the best for specimens containing a low number of microorganisms of interest.

We evaluated aliquots from 244 clinical blood culture bottles that demonstrated yeasts on Gram stain using a Candida albicans peptide nucleic acid (PNA) fluorescent in situ hybridization (FISH) probe. The sensitivity, specificity, positive predictive value, and negative predictive value of the C. albicans PNA FISH test in this study were 99%, 100%, 100%, and 99.3%, respectively.

The bacteria associated with orthopaedic infections are usually common gram-positive and gram-negative bacteria. This fundamental grouping of bacteria is a necessary first step in the selection of appropriate antibiotics. Since polymerase chain reaction (PCR) is more rapid and may be more sensitive than culture, we developed a postamplification pyrosequencing method to subcategorize bacteria based on a few nucleotide polymorphisms in the 16S rRNA gene. We validated this method using well-characterized strains of bacteria and applied it to specimens from spinal surgery cases with suspected infections. Lysates of 114 bacteria including 75 species were created following standard cultivation to obtain DNA. The DNA was amplified by a broad-range real-time PCR. The amplicons were evaluated by pyrosequencing and were classified as gram-positive, gram-negative, or acid-fast bacilli based on the first three to five nucleotides sequenced. In addition, clinical cases of suspected infection were obtained from spinal surgery. The results of the "molecular Gram stain" were compared with the results of traditional Gram stain and culture. The lysates of 107 (93.9%) of the bacteria extracts tested were appropriately categorized as gram-positive and gram-negative or as acid-fast bacilli on the basis of this assay. The sensitivity and specificity of this assay were 100% and 97.4% for gram-positive and 88.3% and 100% for gram-negative isolates. All of the five clinical samples were appropriately categorized as containing gram-positive or gram-negative bacteria with this assay. This study demonstrates that high sensitivity and specificity of a molecular gram stain may be achieved using broad-range real-time PCR and pyrosequencing.

Pyrosequencing technology, sequencing by addition, was evaluated for categorization of mycobacterial isolates. One hundred and eighty-nine isolates, including 18 ATCC and Trudeau Mycobacterial Culture Collection (TMC) strains, were studied. There were 38 Mycobacterium tuberculosis complex, 27 M. kansasii, 27 MAI complex, 21 M. marinum, 14 M. gordonae, 20 M. chelonae-abscessus group, 10 M. fortuitum, 5 M. xenopi, 3 M. celatum, 2 M. terrae complex, 20 M. mucogenicum, and 2 M. scrofulaceum. Nucleic acid extracts were prepared from solid media or MGIT broth. Traditional PCR was performed with one of the primers biotinylated; the assay targeted a portion of the 16S rRNA gene that contains a hypervariable region, which has been previously shown to be useful for the identification of mycobacteria. The PSQ Sample Preparation Kit was used, and the biotinylated PCR product was processed to a single-stranded DNA template. The sequencing primer was hybridized to the DNA template in a PSQ96 plate. Incorporation of the complementary nucleotides resulted in light generation peaks, forming a pyrogram, which was evaluated by the instrument software. Thirty basepairs were used for isolate categorization. Manual interpretation of the sequences was performed if the quality of the 30-bp sequence was in doubt or if more than 4 bp homopolymers were recognized. Sequences with more than 5 bp of bad quality were deemed unacceptable. When blasted against GenBank, 179 of 189 sequences (94.7%) assigned isolates to the correct molecular genus or group. Ten M. gordonae isolates had more than 5 bp of bad quality sequence and were not accepted. Pyrosequencing of this hypervariable region afforded rapid and acceptable characterization of common, routinely isolated clinical Mycobacterium sp. Algorithms are recommended for further differentiation with an additional sequencing primer or additional biochemicals.

We describe broad-range salmonellae (ie, Salmonella) and Salmonella serotype Typhi-specific LightCycler (Roche Diagnostics, Indianapolis, IN) real-time polymerase chain reaction assays. We validated these with a battery of 280 bacteria, 108 of which were salmonellae representing 20 serotypes. In addition, 298 isolates from 170 clinical specimens that were suspected to possibly represent Salmonella were tested with the pan- Salmonella assay. Finally, the pan-Salmonella assay also was used to test DNA extracts from 101 archived, frozen stool specimens, 55 of which were culture-positive for salmonellae. Both assays were 100% sensitive and specific when cultured isolates of the battery were tested. The pan- Salmonella assay also characterized correctly all salmonellae on the primary isolation agar and was 96% sensitive (53/55) and 96% specific (49/51) when nucleic acid extracts from direct stool specimens were tested. These assays represent potential tools the clinical microbiologist could use to screen suspect isolates or stool specimens for Salmonella.

The utility of cytomegalovirus (CMV) viral load (VL) by quantitative hybrid capture assay (Q-HCA) was investigated in bronchoalveolar lavage (BAL) from lung transplant recipients and compared with BAL cultures and blood VL. Forty-three consecutive BAL samples from 27 lung transplant recipients were analyzed. All samples had shell vial (SV) cultures in addition to Q-HCA. Histopathology was done on all lung tissues, and immunohistochemistry (IHC) in those with positive CMV cultures. Fifteen (56%) lung transplant recipients had both positive BAL SV cultures and BAL VL. Five of 15 had CMV pneumonitis with a VL in BAL >500 000 copies/mL (mean: 1638 450). Ten patients without CMV pneumonitis had VL in BAL <500 000 copies/mL (mean 81 820, p = 0.002). High VL in BAL and blood invariably meant CMV pneumonitis, but 2 patients with CMV pneumonitis had high BAL VL but relatively low blood VL. Initial CMV seronegativity was associated with pneumonitis (4/5 vs. 1/10; p = 0.004) and higher BAL CMV VL (p = 0.03). High CMV BAL or blood VL did not correlate with acute rejection or development of bronchiolitis obliterans syndrome (BOS). High CMV VL in BAL in lung transplant recipients is strongly associated with CMV pneumonitis, and may be more predictive than peripheral blood viral load.

We sought to determine if the BK and JC polyomaviruses were associated with idiopathic pulmonary fibrosis (IPF). We did not detect the BK or JC polyomaviruses in lung tissue extracts from 33 patients with IPF by using real-time PCR, which suggests that an etiologic association is unlikely.

The efficiencies of five commercially available nucleic acid extraction methods were evaluated for the recovery of a standardized inoculum of Legionella pneumophila in respiratory specimens (sputum and bronchoalveolar lavage [BAL] specimens). The concentrations of Legionella DNA recovered from sputa with the automated MagNA Pure (526,200 CFU/ml) and NucliSens (171,800 CFU/ml) extractors were greater than those recovered with the manual methods (i.e., Roche High Pure kit [133,900 CFU/ml], QIAamp DNA Mini kit [46,380 CFU/ml], and ViralXpress kit [13,635 CFU/ml]). The rank order was the same for extracts from BAL specimens, except that for this specimen type the QIAamp DNA Mini kit recovered more than the Roche High Pure kit.

A real-time PCR assay that uses two fluorescence resonance energy transfer probe sets and targets the tuf gene of staphylococci is described here. One probe set detects the Staphylococcus genus, whereas the other probe set is specific for Staphylococcus aureus. One hundred thirty-eight cultured isolates, which contained 41 isolates of staphylococci representing at least nine species, and 100 positive blood cultures that contained gram-positive cocci in clusters were tested. This assay was 100% sensitive and 100% specific for the detection of the Staphylococcus genus and of S. aureus.

More yeasts and molds are now recognized to cause more human disease than ever before. This development is not due to a change in the virulence of these fungi, but rather to changes in the human host. These changes include immunosuppression secondary to the pandemic of HIV, the use of life-saving advances in chemotherapy and organ transplantation, and the use of corticosteroids and other immunosuppressive agents to treat a variety of diseases. Fungi that were once considered common saprophytes are now recognized as potential pathogens in these patients. This situation necessitates better communication than ever between the clinician, pathologist, and clinical mycologist to ensure the prompt and accurate determination of the cause of fungal diseases.

We examined four staining methods on replicate smears of 313 respiratory specimens submitted for Pneumocystis jiroveci examination. The sensitivity and specificity of Calcofluor white stain (CW) were 73.8 and 99.6%, respectively. The sensitivity and specificity of Grocott-Gomori methenamine silver stain (GMS) were 79.4 and 99.2%, respectively. The sensitivity and specificity of Diff-Quik stain were 49.2 and 99.6%, respectively. The sensitivity and specificity of Merifluor Pneumocystis stain were 90.8 and 81.9%, respectively. Only CW and GMS had positive and negative predictive values of >90%.

The use of humanized antibody against tumor necrosis factor alpha (TNF-alpha) may increase the risk of various opportunistic infections, including tuberculosis and fungal infections. We report a case of cryptococcal pneumonia in a patient who was taking infliximab for rheumatoid arthritis. A temporally related exposure history raised the possibility that our patient acquired the infection from his pet cockatiel. It seems prudent to advise patients receiving infliximab to avoid exposure to pet avian excreta.

Cytomegalovirus (CMV) is an important pathogen in lung transplant recipients. Early detection of CMV end-organ disease should help with treatment management. We determined the CMV viral load by hybrid capture in bronchoalveolar lavage (BAL) fluid samples from patients who had undergone lung transplantation. For 39 of these samples (from 25 patients), corresponding transbronchial biopsy samples were available for CMV immunohistochemistry (IHC). The CMV IHC results were interpreted and categorized as positive or negative, and the positive results were subcategorized as typical if cells with both significant nuclear enlargement or Cowdry A-type inclusions and positive staining were present or as atypical if definitive nuclear staining was seen but significant nuclear enlargement was not. Diagnostic CMV viral inclusions were reported in the anatomic diagnosis, based on hematoxylin-eosin staining alone, for three (8%) of the biopsy samples. CMV was detected by IHC in 13 (33%) samples (5 typical, 8 atypical). The median CMV viral load in BAL samples was 0 copies/ml for BAL samples from patients with IHC-negative biopsy samples; 47,678 copies/ml for BAL samples from patients with biopsy samples with positive, atypical staining; and 1,548,827 copies/ml for BAL samples from patients with biopsy samples with positive, typical staining (P < 0.001). Compared to routine pathology of biopsy samples, the use of IHC increased the diagnostic yield of CMV. Also, the CMV viral load in BAL fluid samples increased along with immunoreactivity from negative to positive, atypical staining to positive, typical staining. The CMV viral load determined with the end-organ sample, the BAL fluid sample, was higher than the corresponding viral load determined with blood. Both IHC and determination of the CMV viral load in BAL samples may be useful for the detection of individuals at risk for the development of fulminant invasive CMV disease.

Background: Despite existing clinical and histopathological evidence, the role of inflammation in the pathogenesis of inverted papilloma (IP) is not well understood The goal of this study was to describe the inflammatory cell component present in sinonasal papilloma (SP), with the intention of further defining the existence of inflammation in IP and perhaps gaining insight into IP pathophysiology., Methods: Computerized database analysis was performed to identify all patients with SP who underwent surgery at the Cleveland Clinic Foundation between 1995 and 2001. Histopathological features of all SP were reviewed and semiquantitative analysis of the inflammatory cells present was performed. IP was histopathologically graded into four categories by the extent of inflammatory infiltrate and cellular atypia. Statistical analysis of the inflammatory cell component present in the epithelial layer of exophytic papilloma and IP was performed., Results: SP was classified into three types: cylindrical papilloma (5% [2/41]), exophytic squamous papilloma (34% [14/ 41]), and IP (61% [25/41]). Twenty-eight instances of IP in 25 patients were identified. Altogether, 11% were grade I (3/28), 54% were grade II (15/28), 25% were grade III (7/28), and 11% were grade IV (3/28). The inflammatory cell population was significantly greater in IP compared with other SPs and greater in grades I and II IP when compared with grade III and IV IP., Conclusion: Inflammatory cells were identified as a significant cell population in IP, whereas it was less commonly encountered in other forms of SP. The proposed IP staging system may serve as the foundation for improved understanding of IP and, ultimately, may help to predict recurrence or apparent malignant transformation.

We evaluated 2 methods, a LightCycler PCR assay and pyrosequencing for the detection of the JC polyoma virus (JCV) in fixed brain tissue of 10 patients with and 3 control patients without progressive multifocal leukoencephalopathy (PML). Nucleic acid extraction was performed after deparaffinization and proteinase K digestion. The LightCycler assay differentiates the BK virus (BKV), JCV, and SV40 using melt curve analysis. Conventional PCR was used with the same primers to generate products for pyrosequencing. Two sequencing primers were used that differentiate the polyoma viruses. Seven of 11 biopsies (1 patient had 2 biopsies) with PML were positive for JCV by real-time PCR and/or PCR/pyrosequencing. Three of 4 remaining biopsies were positive by real-time PCR but had melting points between JCV and SV40. The 4 specimens that were negative or atypical by LightCycler PCR were positive by traditional PCR, but 1 had an amplicon of lower molecular weight by gel electrophoresis. These were shown to represent JCV by at least 1 of the 2 pyrosequencing primers. The biopsies from patients without PML were PCR negative. Both the LightCycler and pyrosequencing assays are useful for confirming JCV in brain biopsies from patients with PML, but variant JCVs may require supplementary methods to confirm JCV infection.

Susceptibility profiles of medically important fungi in less-developed countries remain uncharacterized. We measured the MICs of amphotericin B, 5-flucytosine, fluconazole, itraconazole, and ketoconazole for Cryptococcus neoformans clinical isolates from Thailand, Malawi, and the United States and found no evidence of resistance or MIC profile differences among the countries.

Mycobacteria cause a variety of illnesses that differ in severity and public health implications. The differentiation of Mycobacterium tuberculosis from nontuberculous mycobacteria (NTM) is of primary importance for infection control and choice of antimicrobial therapy. Despite advances in molecular diagnostics, the ability to rapidly diagnose M. tuberculosis infections by PCR is still inadequate, largely because of the possibility of false-negative reactions. We designed and validated a real-time PCR for mycobacteria by using the LightCycler system with 18 reference strains and 168 clinical mycobacterial isolates. All clinically significant mycobacteria were detected; the mean melting temperatures (with 99.9% confidence intervals [99.9% CI] in parentheses) for the different mycobacteria were as follows: M. tuberculosis, 64.35 degrees C (63.27 to 65.42 degrees C); M. kansasii, 59.20 degrees C (58.07 to 60.33 degrees C); M. avium, 57.82 degrees C (57.05 to 58.60 degrees C); M. intracellulare, 54.46 degrees C (53.69 to 55.23 degrees C); M. marinum, 58.91 degrees C (58.28 to 59.55 degrees C); rapidly growing mycobacteria, 53.09 degrees C (50.97 to 55.20 degrees C) or 43.19 degrees C (42.19 to 44.49 degrees C). This real-time PCR assay with melting curve analysis consistently accurately detected and differentiated M. tuberculosis from NTM. Detection of an NTM helps ensure that the negative result for M. tuberculosis is a true negative. The specific melting temperature also provides a suggestion of the identity of the NTM present, when the most commonly encountered mycobacterial species are considered. In a parallel comparison, both the LightCycler assay and the COBAS Amplicor M. tuberculosis assay correctly categorized 48 of 50 specimens that were proven by culture to contain M. tuberculosis, and the LightCycler assay correctly characterized 3 of 3 specimens that contained NTM.

Background: Identification of the agents of infectious diarrhea may facilitate appropriate therapy and prevent inappropriate antibiotic use., Objectives: To better define the etiology of infectious diarrhea for children <12 years in our community and to study the ordering patterns of physicians., Study Design: We reviewed test results of stool specimens from children <12 years old at our institution (CCF) and those submitted through our reference laboratory for rotavirus enzyme immunoassay (REIA) and stool cultures for a 7-month period (11/1/00-6/1/01). For CCF patients, REIA and stool cultures for usual bacterial enteric pathogens (BEP) were performed, regardless of the test ordered (i.e. REIA alone, stool culture alone or both). We compared the results with the orders placed to determine if requests for rotavirus alone or bacterial stool culture alone missed BEP or rotavirus, respectively., Results: Overall, REIAs were performed on 81% (538/661) of stool specimens, with 37% positive. Stool cultures were performed on 62% (408/661) of stool specimens, with 4.4% positive. Stool specimens (280) from CCF pediatric patients were evaluated for both rotavirus and BEP. Some 42% of REIA and 23% of stool cultures were ordered as single tests, while both tests were ordered for 35% of the patients. Of the REIA ordered alone, 34% were positive for rotavirus; however, 2.5% of these contained BEP that would have been missed. Of the stool cultures that were ordered alone, 8% were positive; however, 19% of these contained rotavirus that would have been missed. When both tests were ordered, 22% contained rotavirus and 2% contained BEP., Conclusion: Both rotavirus and bacterial enteric infections were missed with selective viral versus bacterial specific ordering patterns. A rotaviral screen prior to stool culture may be useful for children with diarrhea during the winter months.

Although rare, pyogenic liver abscesses are potentially fatal. We evaluated the predictive value of Gram stain of liver abscess aspirates and temporally associated blood cultures. Gram stains detected bacteria in 79% of the liver abscesses tested. The sensitivity and specificity of Gram stain of the liver abscesses were 90% and 100% for Gram-positive cocci (GPC) and 52% and 94% for Gram-negative bacilli (GNB). The sensitivities of the blood cultures for any GPC and GNB present in the liver abscess were 30% and 39%, respectively. Although, Gram stains and blood cultures offer incomplete detection of the microbial contents of pyogenic liver abscesses, both tests should always accompany liver abscess cultures.

Extended spectrum beta-lactamases are modified beta-lactamase enzymes that impart resistance to third-generation cephalosporins and make all beta-lactam antibiotics and cephalosporins useless for therapy. We compared the antimicrobial susceptibility profiles of extended-spectrum beta-lactamase (ESBL)-producing and non-ESBL-producing isolates of Klebsiella pneumoniae. The ESBL producers had significantly diminished susceptibility compared with the non-ESBL producers for gentamicin (P < .001), tobramycin (P < .001), amikacin (P < .005), trimethoprim-sulfamethoxazole (P < .01), ciprofloxacin (P < .001), and nitrofurantoin (P < .001). All isolates were susceptible to imipenem. ESBL-producing K pneumoniae may also be resistant to non-beta-lactam antibiotics. Therefore, susceptibility testing of these isolates is critical for guiding therapy.

North American paragonimiasis is well described in omnivorous and carnivorous animals on this continent. Humans are rarely infected, largely because of dietary customs, but are at risk for infection if raw or undercooked crayfish are consumed. We describe a patient with a pleuropulmonary infection due to Paragonimus kellicotti that presented as recurrent pneumothoraces and a cavitary lesion. This is the first case of North American paragonimiasis in which the diagnosis was based on the morphology of the eggs present in histologic sections.

Purpose: The multitarget fluorescence in situ hybridization (FISH) probe set UroVysion (Vysis, Downers Grove, Illinois), containing probes to chromosomes 3, 7 and 17, and to the 9p21 band, has been recently shown to have high sensitivity and specificity for detecting transitional cell carcinoma. In this study we retrospectively tested 120 urine samples from patients with atypical, suspicious and negative cytology for whom concurrent and followup bladder biopsy data were available. We evaluated the ability of FISH to identify malignant cells in cytologically equivocal or negative cases., Materials and Methods: Archived slides from 120 voided (47) or instrumented (73) urine cytology specimens from patients with concurrent bladder biopsy and a minimum of 12 months of biopsy followup were subjected to hybridization with UroVysion. The cohort included patients with biopsy proven transitional cell carcinoma, which was grades 1 to 3 in 23, 35 and 24, respectively, and stages pTis in 3, pTa in 64, pT1 in 6, pT2 in 6 and pT4 in 3, while it showed negative histology in 38. Cytology findings were suspicious, atypical and negative for transitional cell carcinoma in 31, 49 and 40 cases, respectively. A positive FISH result was defined as 5 transitional cells or greater with a gain of 2 or more of chromosomes 3, 7 or 17, 12 cells or greater with 9p21 deletion, or 10% or greater of cells with isolated trisomy of 1 of chromosomes 3, 7 and 17., Results: All except 12 of the 82 biopsy proven transitional cell carcinoma cases (11 pTa and 1 pT1 tumors) were positive by FISH (85% sensitivity). Sensitivity in patients with suspicious, atypical and negative cytology was 100%, 89% and 60%, respectively. Nine patients with atypical cytology had positive FISH in the setting of a negative concurrent bladder biopsy. However, 8 of these 9 patients (89%) had biopsy proven transitional cell carcinoma within 12 months following the date when the sample tested by FISH was obtained. The last of these patients with false-positive results had previously documented pTis disease, which was also present in the next bladder biopsy 15 months following the positive FISH result. The remaining 29 specimens from patients with negative biopsy and a negative 12-month followup tested negative by FISH (97% overall specificity)., Conclusions: The UroVysion FISH assay provides high sensitivity and specificity to detect transitional cell carcinoma in cytologically equivocal and negative urine samples. These results emphasize the important role of this assay in the management of bladder cancer.

Objective: To determine the accuracy and cost-effectiveness of a polymerase chain reaction (PCR) for detecting nasal carriage of Staphylococcus aureus directly from clinical specimens. CROSS-SECTIONAL STUDY: This occurred in a tertiary-care hospital in Cleveland, Ohio, and included 239 consecutive patients who were scheduled for a cardiothoracic surgical procedure. Conventional cultures and a PCR for S. aureus from nasal swabs were used as measurements. COST-EFFECTIVENESS ANALYSIS: Data sources were market prices and Bureau of Labor Statistics. The time horizon was the maximum period for availability of culture results (3 days). Interventions included universal mupirocin therapy without testing; initial therapy, with termination if PCR negative (treat-PCR); initial therapy, with termination if culture negative (treat-culture); treat PCR-positive carriers (PCR-guided treatment); and treat culture-positive carriers (culture-guided treatment). The perspective was institutional and costs and the length of time to treatment were outcome measures., Results: Sixty-seven (28%) of the 239 swabs grew S. aureus. Rapid PCR was 97.0% sensitive and 97.1% specific for the detection of S. aureus. For populations with prevalences of nasal S. aureus carriage of up to 50%, the PCR assay had negative predictive values of greater than 97%. PCR-guided treatment had the lowest incremental cost-effectiveness ratio (1.93 dollars per additional day compared with the culture strategy). Among immediate treatment strategies, treat-PCR was most cost-effective. The universal therapy strategy cost 38.19 dollars more per additional day gained with carrier identification compared with the PCR strategy., Conclusion: Rapid real-time PCR is an accurate, rapid, and cost-effective method for identifying S. aureus carriers for preoperative intervention.

Antimicrobial resistance among bacterial pathogens is a global problem, but in Egypt data are sparse. We reviewed the antimicrobial susceptibility patterns of bloodstream isolates of Gram-positive cocci and Gram-negative bacilli in five hospitals in Cairo, Egypt, from 1999 to 2000. In addition, susceptibilities of non-bloodstream isolates of Streptococcus pneumoniae and Enterococcus spp. were analysed. High rates of resistance were found in most of the bacteria studied. In the hospitals, a variety of methods were used for identification and susceptibility testing, but in the laboratories quality controlled strains were utilized routinely, to ensure accurate performance of the assays. Only 29% of Staphylococcus aureus isolates and 23% of coagulase-negative staphylococcal isolates were oxacillin susceptible. Both groups of staphylococci were also highly resistant to erythromycin, co-trimoxazole, clindamycin and doxycycline; all isolates were susceptible to vancomycin. Susceptibility of S. pneumoniae isolates to penicillin, ceftriaxone and fluoroquinolones was 63%, 84% and 82%, respectively. Vancomycin susceptibility of the enterococci was 96%; susceptibility to high-level gentamicin and streptomycin was 54% and 48%, respectively. Resistance to most relevant antimicrobials was commonplace among the Gram-negative bacilli; however, most remained susceptible to imipenem. The percentage of bloodstream isolates of Escherichia coli susceptible to common antimicrobial agents was as follows: ampicillin (6%), ampicillin-sulbactam (38%), co-trimoxazole (38%) and aminoglycosides (52%). The susceptibility of isolates of E. coli, Klebsiella and Enterobacter spp. to ceftazidime was 62%, 40% and 46%, respectively. This suggests a potentially high rate of extended-spectrum beta-lactamase (ESBL) and/or Amp-C enzyme production. These results call for a nationwide surveillance programme to monitor microbial trends and antimicrobial resistance patterns in Egypt.

A retrospective chart review of all patients seen at the Cleveland Clinic Foundation with infectious endogenous endophthalmitis between January 1982 and August 2000 revealed 34 affected eyes in 27 patients. During this time, the median incidence of endogenous endophthalmitis was 1.8 cases/year, and 48.1% of patients presented as outpatients. Twenty-six patients presented to an ophthalmologist, and the diagnosis was initially missed in almost half the cases. Eleven patients had an unremarkable physical exam except for eye findings. We found an equal incidence of bacterial and fungal endophthalmitis and a predominance of among the fungal etiologic agents. We did not, however, note a predominance of Gramnegative organisms seen mostly in reports from Asia. The microbiologic diagnosis was based on aqueous and vitreous cultures or positive eye histopathology stains in almost two-thirds of cases. The sensitivity of the Gram stain was poor, but its specificity and positive predictive value were excellent. The vitreous cultures obtained by vitrectomy instruments were more sensitive in making the diagnosis than the vitreous needle biopsy. Aside from blood cultures and eye specimen cultures, half the patients had an additional infectious focus, most frequently a urinary tract infection, whereas infectious endocarditis was seen in a small minority. Twelve patients had visual improvement with treatment with a final visual acuity better than 20/200 in 44% of the eyes. Good visual outcome was associated with visual acuity of 20/200 or better at diagnosis and with the absence of hypopyon.

We designed and tested a real-time LightCycler PCR assay for Histoplasma capsulatum that correctly identified the 34 H. capsulatum isolates in a battery of 107 fungal isolates tested and also detected H. capsulatum in clinical specimens from three patients that were culture positive for this organism.

We describe a patient with a urinary tract infection (UTI) caused by Pasteurella multocida. Pulsed-field gel electrophoresis revealed that the clinical isolate recovered from the patient was identical (100% band match) to P. multocida isolates recovered from the patient's cat, but the isolate differed from an isolate recovered from a visiting cat and from a laboratory control strain. The patient also had abnormal urologic anatomy secondary to surgery; this has also been associated with P. multocida UTI.

Fluorescence in situ hybridization (FISH) using peptide nucleic acid (PNA) probes targeting Staphylococcus aureus 16S rRNA is a novel method for direct identification of S. aureus from positive blood culture bottles. The test (S. aureus PNA FISH) is performed on smears made directly from positive blood culture bottles with gram-positive cocci in clusters (GPCC) and provides results within 2.5 h. A blinded comparison of S. aureus PNA FISH with standard identification methods was performed in collaboration with eight clinical microbiology laboratories. A total of 564 routine blood culture bottles positive for GPCC recovered from both aerobic and anaerobic media from three different manufacturers (ESP, BACTEC, and BacT/Alert) were included in the study. The sensitivity and specificity of S. aureus PNA FISH were 100% (57 of 57) and 99.2% (116 of 117), respectively, with 174 GPCC-positive ESP blood culture bottles, 98.5% (67 of 68) and 98.5% (129 of 131), respectively, with 200 GPCC-positive BACTEC blood culture bottles, and 100% (74 of 74) and 99.1% (115 of 116), respectively, with 190 GPCC-positive BacT/Alert blood culture bottles. It is concluded that S. aureus PNA FISH performs well with commonly used continuously monitoring blood culture systems.

The increasing prevalence of vancomycin-resistant enterococcal (VRE) infections has necessitated a search for drugs that are effective in treating these infections, and a need to determine whether currently available antimicrobials are effective. 75 consecutive clinical isolates of vancomycin-resistant Enterococcus faecium (VRE faecium) (40 blood and 35 urine isolates) isolated over 1 y at the Cleveland Clinic Foundation were tested for susceptibility to linezolid, quinupristin-dalfopristin, fosfomycin and nitrofurantoin using the Etest. The minimum inhibitory concentrations were read independently by 3 observers and compared, and a final reading was obtained by predetermined criteria. The proportion of isolates susceptible to linezolid, quinupristin-dalfopristin, fosfomycin and nitrofurantoin was 100%, 98.7%, 98.7% and 78.7%, respectively. No single isolate was resistant to more than 1 of the 4 drugs tested. Etest presented significant unexpected difficulties in testing for VRE faecium susceptibility to nitrofurantoin. Fosfomycin may be a useful alternative to linezolid and quinupristin-dalfopristin in the treatment of VRE infections in certain clinical situations, e.g. uncomplicated urinary tract infections. In addition, the use of fosfomycin could limit the use of newer agents, thus reducing the chance of development of further resistance in the enterococci.