Evaluation of the BioFire FilmArray® GastrointestinalPanel in a Midwestern Academic Hospital.

The BioFire FilmArray® Gastrointestinal Panel (GIP) was implemented to replace traditional stool culture and enzyme immunoassay (EIA) testing for stool pathogens. The purpose of this study was to evaluate the detection rate, incidence of coinfection, and culture recovery rate of gastrointestinal (GI) pathogens detected by the GIP over a 1-year period. A total of 2257 stools collected from January to December 2015 were tested using the GIP. Clostridium difficile colonization was also evaluated by an antigen/toxin EIA and confirmatory polymerase chain reaction (PCR). The GIP detected one pathogen in 911 (40.4%) specimens. Coinfections were detected in 176 (7.8%) of these specimens. The most frequently detected pathogens were C. difficile (15.2%), norovirus (8.9%), enteropathogenic Escherichia coli (7.1%), enteroaggregative E. coli (3.4%), Campylobacter spp. (2.3%), and sapovirus (2.0%). Each of the remaining GIP targets had a detection rate of ≤1.6%. The recovery of bacteria for public health investigations varied, with rates as high as 77% for Salmonella to as low as 30% for Yersinia enterocolitica. Of stools positive for C. difficile on the GIP that were tested by EIA, only 42.7% (88/206) were found to be producing detectable toxin. Overall, the implementation of the GIP resulted in high detection rates of GI pathogens, including the frequent detection of coinfections. This is a promising test to streamline the testing of agents causing infectious gastroenteritis from multiple tests down to a single order with limited hands-on time. Ongoing studies will need to assess the impact that the GIP has on downstream patient care and public health practices.

Assessment of whole-genome mapping in a well-defined outbreak of Salmonella enterica serotype Saintpaul.

We investigated the use of whole-genome mapping and pulsed-field gel electrophoresis (PFGE) with isolates from an outbreak of Salmonella enterica serotype Saintpaul. PFGE and whole-genome mapping were concordant with 22 of 23 isolates. Whole-genome mapping is a viable alternative tool for the epidemiological analysis of Salmonella food-borne disease investigations.

Paecilomyces lilacinus infection in a liver transplant patient: case report and review of the literature.

A 56-year-old male who was 12 months status post liver transplant presented with a 2-month history of painful, erythematous nodules over the right knee. Several biopsies yielded a mold initially phenotypically identified as a Penicillium species, but molecular sequence analysis ultimately determined the identity as Paecilomyces lilacinus. Several courses of oral voriconazole were required for resolution of the infection. A review of the literature revealed that Paecilomyces species are an infrequent cause of disease in transplant patients, with skin and soft tissue infections being the most common presentation. It is important to accurately identify these infections, and polymerase chain reaction assay using universal fungal primers offers a rapid and precise diagnostic approach. Treatment of Paecilomyces infections may require multiple courses of antifungal therapy, often with surgical debridement. We suggest that voriconazole may be a useful treatment alternative to the more traditional therapy with amphotericin B-based agents.

Molecular analysis of Francisella tularensis subspecies tularensis and holarctica.

Rapid methods are needed for public health and military applications to specifically identify Francisella tularensis, the causative agent of tularemia in humans. A comparative analysis of the capabilities of multiple technologies was performed using a well-defined set of organisms to determine which approach would provide the most information in the shortest time. High-resolution molecular techniques, including pulsed-field gel electrophoresis, amplified fragment length polymorphism, and ribotyping, provided subspecies level identification within approximately 24 hours after obtaining an isolate, whereas multilocus variable number tandem repeat analysis with 8 or 25 targets provided strain level discrimination within about 12 hours. In contrast, Raman spectroscopy provided species level identification in 10 minutes but could not differentiate between subspecies tularensis and holarctica.

Genomic deletion marking an emerging subclone of Francisella tularensis subsp. holarctica in France and the Iberian Peninsula.

Francisella tularensis subsp. holarctica is widely disseminated in North America and the boreal and temperate regions of the Eurasian continent. Comparative genomic analyses identified a 1.59-kb genomic deletion specific to F. tularensis subsp. holarctica isolates from Spain and France. Phylogenetic analysis of strains carrying this deletion by multiple-locus variable-number tandem repeat analysis showed that the strains comprise a highly related set of genotypes, implying that these strains were recently introduced or recently emerged by clonal expansion in France and the Iberian Peninsula.

Identification and first report of Inonotus (Phellinus) tropicalis as an etiologic agent in a patient with chronic granulomatous disease.

Although isolates of filamentous basidiomycetes can usually be recognized in a clinical laboratory setting, identification is problematic, as they seldom exhibit diagnostic morphological features formed in nature. This paper is the first report of Inonotus (Phellinus) tropicalis inciting human disease and describes the methods used to support the identification.

Histoplasmosis in solid organ transplant recipients at a large Midwestern university transplant center.

Histoplasma capsulatum sporadically causes severe infections in solid organ transplant (SOT) patients in the Midwest, but it has been an unusual infection among those patients followed at the University of Nebraska Medical Center (UNMC), located at the western edge of the 'histo belt.' Nine SOT patients with histoplasmosis are described (6 renal or renal-pancreas and 3 liver recipients) who developed severe histoplasmosis over a recent 2.5-year period at UNMC. Symptoms started a median of 11 months (range, 1.2-90 months) after organ transplant and consisted primarily of fever, cough, shortness of breath, and malaise or fatigue present for approximately 30 days prior to medical evaluation. All patients had an abnormal chest radiograph and/or computed tomographic scan. Tacrolimus was the main immunosuppressant in all 9 patients, along with prednisone or mycophenolate. Dacluzimab or thymoglobulin had been given around the time of transplant in 6 of 9. None was treated for an episode of acute rejection within 2 months before onset of histoplasmosis, although 2 were on high-dose immunosuppression after recent transplants. Diagnosis was made by culture in 8 of the 9 patients, with positive serum and urine histoplasma antigen tests in all 9 cases. From 1997 to 2001, during a period of relative quiescence of the disease in the general population, the rate of clinical histoplasmosis among SOT patients at UNMC was estimated at 0.11%, whereas during 2002 through the first half of 2004, the rate rose 17-fold to 1.9%. Histoplasmosis can present as a prolonged febrile illness with subacute pulmonary symptoms in a cohort of SOT patients, despite the absence of a regional outbreak.

Genome diversity among regional populations of Francisella tularensis subspecies tularensis and Francisella tularensis subspecies holarctica isolated from the US.

Francisella tularensis is a highly infectious facultative intracellular pathogen that is considered a potential agent of bioterrorism. Four different F. tularensis subspecies have been identified and they appear to display different ecological and virulence characteristics as well as differences in geographical distribution. One simple explanation for the variation in ecological and virulence characteristics is that they are conferred by differences in genome content. To characterize genome content among stains isolated from United States, we have used a DNA microarray designed from a shotgun library of a reference strain. Polymorphisms distributed among polyphyletic sets of strains was the most common pattern of genome alteration observed, indicating that strain-specific genome variability is significant. Nonetheless, 13 different contiguous segments of the genome were found to be missing exclusively in each of the subsp. holarctica strains tested. All 13 are associated with repeat sequences or transposases that could promote insertion/deletion events. Comparison of the live vaccine strain to other holarctica strains also identified three regions that are absent exclusively in the live vaccine strain derived from holarctica.

Utilization of the internal transcribed spacer regions as molecular targets to detect and identify human fungal pathogens.

Advances in molecular technology show great potential for the rapid detection and identification of fungi for medical, scientific and commercial purposes. Numerous targets within the fungal genome have been evaluated, with much of the current work using sequence areas within the ribosomal DNA (rDNA) gene complex. This section of the genome includes the 18S, 5.8S and 28S genes which code for ribosomal RNA (rRNA) and which have a relatively conserved nucleotide sequence among fungi. It also includes the variable DNA sequence areas of the intervening internal transcribed spacer (ITS) regions called ITS1 and ITS2. Although not translated into proteins, the ITS coding regions have a critical role in the development of functional rRNA, with sequence variations among species showing promise as signature regions for molecular assays. This review of the current literature was conducted to evaluate clinical approaches for using the fungal ITS regions as molecular targets. Multiple applications using the fungal ITS sequences are summarized here including those for culture identification, phylogenetic research, direct detection from clinical specimens or the environment, and molecular typing for epidemiological investigations. The breadth of applications shows that ITS regions have great potential as targets in molecular-based assays for the characterization and identification of fungi. Development of rapid and accurate amplification-based ITS assays to diagnose invasive fungal infections could potentially impact care and improve outcome for affected patients.

Outbreak of vancomycin-resistant Enterococcus faecium in a neonatal intensive care unit.

An outbreak of vancomycin-resistant Enterococcus faecium involving 28 infants in a neonatal intensive care unit was observed. Successful control of the outbreak was achieved following use of patient and staff cohorting, contact isolation precautions, patient and environmental surveillance cultures, environmental decontamination, molecular typing, introduction of an alcohol-based hand disinfectant, and decreased use of vancomycin.

Cutaneous infection caused by Cylindrocarpon lichenicola in a patient with acute myelogenous leukemia.

Cylindrocarpon lichenicola is a saprophytic soil fungus which has rarely been associated with human disease. We report the first case of localized invasive cutaneous infection caused by this fungus in a 53-year-old male from the rural midwestern United States with relapsed acute myelogenous leukemia. On admission for induction chemotherapy, the patient was noted to have an abrasive laceration between the fourth and fifth metacarpophalangeal joints and on the dorsum of the right hand, which progressed to frank ulceration following chemotherapy. A biopsy provided an initial diagnosis of an invasive fungal infection consistent with aspergillosis based on the histopathological appearance of the mold in tissue. Multiple positive fungal cultures which were obtained from the biopsied tissue were subsequently identified by microscopic and macroscopic characteristics to be C. lichenicola. The infection resolved following marrow regeneration, aggressive debridement of the affected tissue, and treatment with amphotericin B. This case extends the conditions associated with invasive disease caused by C. lichenicola.

Ceftriaxone-resistant salmonella infection acquired by a child from cattle.

BACKGROUND: The emergence of resistance to antimicrobial agents within the salmonellae is a worldwide problem that has been associated with the use of antibiotics in livestock. Resistance to ceftriaxone and the fluoroquinolones, which are used to treat invasive salmonella infections, is rare in the United States. We analyzed the molecular characteristics of a ceftriaxone-resistant strain of Salmonella enterica serotype typhimurium isolated from a 12-year-old boy with fever, abdominal pain, and diarrhea. METHODS: We used pulsed-field gel electrophoresis and analysis of plasmids and beta-lactamases to compare the ceftriaxone-resistant S. enterica serotype typhimurium from the child with four isolates of this strain obtained from cattle during a local outbreak of salmonellosis. RESULTS: The ceftriaxone-resistant isolate from the child was indistinguishable from one of the isolates from cattle, which was also resistant to ceftriaxone. Both ceftriaxone-resistant isolates were resistant to 13 antimicrobial agents; all but one of the resistance determinants were on a conjugative plasmid of 160 kb that encoded the functional group 1 beta-lactamase CMY-2. Both ceftriaxone-resistant isolates were closely related to the three other salmonella isolates obtained from cattle, all of which were susceptible to ceftriaxone. CONCLUSIONS: This study provides additional evidence that antibiotic-resistant strains of salmonella in the United States evolve primarily in livestock. Resistance to ceftriaxone, the drug of choice for invasive salmonella disease, is a public health concern, especially with respect to children, since fluoroquinolones, which can also be used to treat this disease, are not approved for use in children.

Identification of Aspergillus species using internal transcribed spacer regions 1 and 2.

Aspergillus species are the most frequent cause of invasive mold infections in immunocompromised patients. Although over 180 species are found within the genus, 3 species, Aspergillus flavus, A. fumigatus, and A. terreus, account for most cases of invasive aspergillosis (IA), with A. nidulans, A. niger, and A. ustus being rare causes of IA. The ability to distinguish between the various clinically relevant Aspergillus species may have diagnostic value, as certain species are associated with higher mortality and increased virulence and vary in their resistance to antifungal therapy. A method to identify Aspergillus at the species level and differentiate it from other true pathogenic and opportunistic molds was developed using the 18S and 28S rRNA genes for primer binding sites. The contiguous internal transcribed spacer (ITS) region, ITS 1-5.8S-ITS 2, from referenced strains and clinical isolates of aspergilli and other fungi were amplified, sequenced, and compared with non-reference strain sequences in GenBank. ITS amplicons from Aspergillus species ranged in size from 565 to 613 bp. Comparison of reference strains and GenBank sequences demonstrated that both ITS 1 and ITS 2 regions were needed for accurate identification of Aspergillus at the species level. Intraspecies variation among clinical isolates and reference strains was minimal. Sixteen other pathogenic molds demonstrated less than 89% similarity with Aspergillus ITS 1 and 2 sequences. A blind study of 11 clinical isolates was performed, and each was correctly identified. Clinical application of this approach may allow for earlier diagnosis and selection of effective antifungal agents for patients with IA.

Pulmonary infection caused by Gymnascella hyalinospora in a patient with acute myelogenous leukemia.

We report the first case of invasive pulmonary infection caused by the thermotolerant ascomycetous fungus Gymnascella hyalinospora in a 43-year-old female from the rural midwestern United States. The patient was diagnosed with acute myelogenous leukemia and treated with induction chemotherapy. She was discharged in stable condition with an absolute neutrophil count of 100 cells per microliter. Four days after discharge, she presented to the Cancer Clinic with fever and pancytopenia. A solitary pulmonary nodule was found in the right middle lobe which was resected by video-assisted thoracoscopy (VATHS). Histopathological examination revealed septate branching hyphae, suggesting a diagnosis of invasive aspergillosis; however, occasional yeast-like cells were also present. The culture grew a mold that appeared dull white with a slight brownish tint that failed to sporulate on standard media. The mold was found to be positive by the AccuProbe Blastomyces dermatitidis Culture ID Test (Gen-Probe Inc., San Diego, Calif.), but this result appeared to be incompatible with the morphology of the structures in tissue. The patient was removed from consideration for stem cell transplant and was treated for 6 weeks with amphotericin B (AmB), followed by itraconazole (Itr). A VATHS with biopsy performed 6 months later showed no evidence of mold infection. In vitro, the isolate appeared to be susceptible to AmB and resistant to fluconazole and 5-fluorocytosine. Results for Itr could not be obtained for the case isolate due to its failure to grow in polyethylene glycol used to solubilize the drug; however, MICs for a second isolate appeared to be elevated. The case isolate was subsequently identified as G. hyalinospora based on its formation of oblate, smooth-walled ascospores within yellow or yellow-green tufts of aerial hyphae on sporulation media. Repeat testing with the Blastomyces probe demonstrated false-positive results with the case isolate and a reference isolate of G. hyalinospora. This case demonstrates that both histopathologic and cultural features should be considered for the proper interpretation of this molecular test and extends the list of fungi recognized as a cause of human mycosis in immunocompromised patients.

Evaluation of the revised MicroScan dried overnight gram-positive identification panel to identify Enterococcus species.

The revised MicroScan Dried Overnight Gram-Positive Identification panel was evaluated for its efficacy at identifying Enterococcus species in comparison with conventional biochemical tests. Supplemental testing of ampicillin-susceptible Enterococcus faecium for motility and the ability to acidify methyl-alpha-D-glucopyranoside helped recognize E. gallinarum and increased the accuracy of the panel for identifying Enterococcus species to 98.5%.

Disseminated aspergillosis caused by Aspergillus ustus in a patient following allogeneic peripheral stem cell transplantation.

The first case of disseminated aspergillosis caused by Aspergillus ustus in an allogeneic peripheral stem cell transplant patient is described. The patient, a 46-year-old female with a history of myelodysplastic syndrome, underwent high-dose chemotherapy and total body irradiation prior to transplantation. She was released from the hospital 49 days posttransplant (p.t.) in a stable condition with an absolute neutrophil count (ANC) of 2,700 cells per microl. Multiple antimicrobial agents, including itraconazole (ITR), were prescribed during hospitalization and at the time of discharge. Three days after discharge, the patient was readmitted with hemorrhagic cystitis, persistent thrombocytopenia, and bilateral pulmonary consolidation, although no fever was present. The ANC at the time of readmission was 3,500. Upon detection of a pulmonary nodule (day 67 p.t.), a bronchoalveolar lavage was performed; the lavage fluid was positive for both cytomegalovirus and parainfluenza virus and negative for fungus. The patient was placed on ganciclovir. A biopsy specimen from a leg lesion also noted on day 67 p.t. revealed septate hyphae consistent with Aspergillus species, and a culture subsequently yielded Aspergillus ustus. Confirmation detection of A. ustus was made by demonstration of characteristic reproductive structures with the presence of Hülle cells. On day 67 p.t., ITR was discontinued and liposomal amphotericin B (AMB) was initiated. The patient's condition worsened, and she died 79 days p.t. At the time of autopsy, septate hyphae were present in heart, thyroid, and lung tissues, with lung tissue culture positive for A. ustus. In vitro susceptibility testing indicated probable resistance to AMB but not to ITR. This case supports the need for the development of rapid methods to determine antifungal susceptibility.

Environmental contamination with vancomycin-resistant enterococci in an outpatient setting.

Eleven cancer patients colonized with vancomycin-resistant enterococci (VRE) were followed as outpatients. Environmental cultures were obtained from clinic rooms before and after patients care. Environmental contamination occurred in 29% of encounters. Superficial disinfection did not eradicate contamination, although more thorough cleaning did. We conclude that environmental VRE contamination occurs in the outpatient setting. Infection control practices, similar to those used in the inpatient setting, may be necessary for outpatient clinics.

Invasive pulmonary aspergillosis due to Aspergillus terreus: 12-year experience and review of the literature.

A 12-year retrospective analysis was done to identify and evaluate in detail cases of invasive pulmonary aspergillosis (IPA) caused by Aspergillus terreus. We identified 13 A. terreus infections among 133 total cases of confirmed invasive aspergillosis; 11 were IPA and 2 were primary peritoneal infections. Of the 11 patients with IPA, 7 developed neutropenia during hospitalization, and the remaining four were receiving immunosuppressive agents. Ten patients with IPA died; one liver transplantation patient without neutropenia survived after treatment with amphotericin B, itraconazole, and a pulmonary lobectomy. Six patients developed disseminated disease, with the heart the most common extrapulmonary site identified (four patients). These cases demonstrate that IPA caused by A. terreus rapidly progresses in immunocompromised patients receiving amphotericin B and illustrate the need for sensitive diagnostic tests and more effective antifungal agents.