Neutrophilic inflammation in gallbladder carcinoma correlates with patient survival: A case-control study.

Gallbladder carcinoma is an uncommon malignancy with an overall 5-year survival of less than 5%. Gallbladder carcinoma has been strongly linked with cholelithiasis and chronic inflammation. Case reports and series have described cholecystitis with acute (neutrophilic) inflammation in association with gallbladder carcinoma, although a clear relationship to patient outcome has not been established. Our series included 8 cases of gallbladder carcinoma with high tumor-associated neutrophils (>25 per high power field) that were associated with shorter patient survival (Cox regression coefficient 6.2, p = 0.004) than age- and stage-matched controls. High tumor-associated neutrophils were not associated with gallbladder rupture/perforation or increased bacterial load measured by 16S PCR. Neutrophilic inflammation with gallbladder carcinoma correlates to shorter survival, independent of patient age and stage of carcinoma. The findings suggest that the degree of neutrophilic inflammation may have prognostic significance in specimens from patients with gallbladder carcinoma after cholecystectomy. Further studies with larger case numbers are needed to confirm and generalize these findings.

Leptotrichia species isolated from a chronic recurrent corneal ulcer.

PURPOSE: To report a case of recurrent corneal ulcer caused by an oropharyngeal cavity pathogen. OBSERVATIONS: A patient presented with recurrent corneal ulcers with hypopyon. Leptotrichia species was eventually isolated from the corneal ulcer on bacterial polymerase chain reaction (PCR) after many negative bacterial culture attempts. Due to correct identification of the pathogen, it was discovered that the patient was exposing her eye to saliva. Modification of patient behavior and initiation of the appropriate antibacterial treatment resulted in resolution of recurrent episodes of active infection. CONCLUSIONS: Although Leptotrichia species are not typically ocular pathogens, they can become pathogenic in the cornea with direct transmission from the oral cavity to the eye.

Back Pain in a 23-Year-Old Male With X-Linked Chronic Granulomatous Disease.

Patients with chronic granulomatous disease are at increased risk for invasive aspergillosis. Cryptic Aspergillus species are being increasingly recognized as distinct causes of infection in this population. In this study, we describe the first case of Aspergillus udagawae vertebral osteomyelitis in a patient with X-linked chronic granulomatous disease.

Seasonal clustering of sinopulmonary mucormycosis in patients with hematologic malignancies at a large comprehensive cancer center.

Background: Invasive Mucorales infections (IMI) lead to significant morbidity and mortality in immunocompromised hosts. The role of season and climatic conditions in case clustering of IMI remain poorly understood. Methods: Following detection of a cluster of sinopulmonary IMIs in patients with hematologic malignancies, we reviewed center-based medical records of all patients with IMIs and other invasive fungal infections (IFIs) between January of 2012 and August of 2015 to assess for case clustering in relation to seasonality. Results: A cluster of 7 patients were identified with sinopulmonary IMIs (Rhizopus microsporus/azygosporus, 6; Rhizomucor pusillus, 1) during a 3 month period between June and August of 2014. All patients died or were discharged to hospice. The cluster was managed with institution of standardized posaconazole prophylaxis to high-risk patients and patient use of N-95 masks when outside of protected areas on the inpatient service. Review of an earlier study period identified 11 patients with IMIs of varying species over the preceding 29 months without evidence of clustering. There were 9 total IMIs in the later study period (12 month post-initial cluster) with 5 additional cases in the summer months, again suggesting seasonal clustering. Extensive environmental sampling did not reveal a source of mold. Using local climatological data abstracted from National Centers for Environmental Information the clusters appeared to be associated with high temperatures and low precipitation. Conclusions: Sinopulmonary Mucorales clusters at our center had a seasonal variation which appeared to be related to temperature and precipitation. Given the significant mortality associated with IMIs, local climatic conditions may need to be considered when considering center specific fungal prevention and prophylaxis strategies for high-risk patients.

Multifocal but non-disseminated phaeohyphomycosis in a healthy man via a unique mechanism: Ejection from motor vehicle accident into a vegetable field in Afghanistan resulting in multiple contaminated skin wounds.

A 20-year-old male presented with multiple subcutaneous nodules on the head, neck, chest and oral cavity. FNA and biopsy showed pigmented fungal hyphae diagnostic of multifocal phaeohyphomycosis, found to be Exophiala spinifera by molecular diagnostics. The presentation initially raised concern for disseminated disease and occult immunosuppression. However, the patient appeared to be immunocompetent and otherwise healthy. Upon further inquiry, the patient was in a motor vehicle accident 4 years before presentation; he was ejected into a vegetable field resulting in multiple open wounds. Multifocal phaeohyphomycosis usually indicates disseminated systemic disease from immunosuppression and carries a grave prognosis.

Incidental identification of Strongyloides stercoralis infection by broad-range 28S rDNA gene sequencing in a patient with a hematolymphoid malignancy.

Strongyloides stercoralis is an important human parasite, especially in rural areas and developing countries. Infected immunosuppressed patients are at risk for hyperinfection, with severe clinical consequences. Here we describe the incidental detection and diagnosis of an unexpected S. stercoralis infection by methods designed to detect fungal 28S ribosomal DNA.

Clinical Next Generation Sequencing Outperforms Standard Microbiological Culture for Characterizing Polymicrobial Samples.

BACKGROUND: Humans suffer from infections caused by single species or more complex polymicrobial communities. Identification of infectious bacteria commonly employs microbiological culture, which depends upon the in vitro propagation and isolation of viable organisms. In contrast, detection of bacterial DNA using next generation sequencing (NGS) allows culture-independent microbial profiling, potentially providing important new insights into the microbiota in clinical specimens. METHODS: NGS 16S rRNA gene sequencing (NGS16S) was compared with culture using (a) synthetic polymicrobial samples for which the identity and abundance of organisms present were precisely defined and (b) primary clinical specimens. RESULTS: Complex mixtures of at least 20 organisms were well resolved by NGS16S with excellent reproducibility. In mixed bacterial suspensions (107 total genomes), we observed linear detection of a target organism over a 4-log concentration range (500-3 × 106 genomes). NGS16S analysis more accurately recapitulated the known composition of synthetic samples than standard microbiological culture using nonselective media, which distorted the relative abundance of organisms and frequently failed to identify low-abundance pathogens. However, extended quantitative culture using selective media for each of the component species recovered the expected organisms at the proper abundance, validating NGS16S results. In an analysis of sputa from cystic fibrosis patients, NGS16S identified more clinically relevant pathogens than standard culture. CONCLUSIONS: Biases in standard, nonselective microbiological culture lead to a distorted characterization of polymicrobial mixtures. NGS16S demonstrates enhanced reproducibility, quantification, and classification accuracy compared with standard culture, providing a more comprehensive, accurate, and culture-free analysis of clinical specimens.

Rapid Detection of Vancomycin-Intermediate Staphylococcus aureus by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry.

Vancomycin is the standard of care for the treatment of invasive methicillin-resistantStaphylococcus aureus(MRSA) infections. Infections with vancomycin-nonsusceptible MRSA, including vancomycin-intermediateS. aureus(VISA) and heterogeneous VISA (hVISA), are clinically challenging and are associated with poor patient outcomes. The identification of VISA in the clinical laboratory depends on standard susceptibility testing, which takes at least 24 h to complete after isolate subculture, whereas hVISA is not routinely detected in clinical labs. We therefore sought to determine whether VISA and hVISA can be differentiated from vancomycin-susceptibleS. aureus(VSSA) using the spectra produced by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Strains of MRSA were characterized for vancomycin susceptibility phenotype by broth microdilution and modified population analysis. We tested 21 VISA, 21 hVISA, and 38 VSSA isolates by MALDI-TOF MS. Susceptibility phenotypes were separated by using a support vector machine (SVM) machine learning algorithm. The resulting model was validated by leave-one-out cross validation. Models were developed and validated by using spectral profiles generated under various subculture conditions, as well as with and without hVISA strains. Using SVM, we correctly identified 100% of the VISA and 97% of the VSSA isolates with an overall classification accuracy of 98%. Addition of hVISA to the model resulted in 76% hVISA identification, 100% VISA identification, and 89% VSSA identification, for an overall classification accuracy of 89%. We conclude that VISA/hVISA and VSSA isolates are separable by MALDI-TOF MS with SVM analysis.

Can anaerobes be acid fast? A novel, clinically relevant acid fast anaerobe.

INTRODUCTION: Anaerobic acid fast bacilli (AFB) have not been previously reported in clinical microbiology. This is the second case report of a novel anaerobic AFB causing disease in humans. CASE PRESENTATION: An anaerobic AFB was isolated from an abdominal wall abscess in a 64-year-old Caucasian diabetic male, who underwent distal pancreatectomy and splenectomy for resection of a pancreatic neuroendocrine tumour. The isolated bacteria were gram-variable and acid-fast, consisting of small irregular rods. The 16S rRNA gene sequence analysis showed that the isolate is a novel organism described in the literature only once before. The organism was studied at the CDC (Centers for Disease Control and Prevention) by the same group that worked with the isolates from the previous report; their findings suggest that the strain belongs to the suborder Corynebacterineae. CONCLUSION: This is the fifth reported case of an anaerobic AFB involved in clinical disease; its microbiological features and 16S RNA sequence are identical to previously reported cases. Clinical disease with this organism seems to be associated with recent history of surgery and abscess formation in deep soft tissues. Acquisition from surgical material is uncertain but seems unlikely.

Bartonella quintana Aortitis in a Man with AIDS, Diagnosed by Needle Biopsy and 16S rRNA Gene Amplification.

A man with newly diagnosed AIDS presented with months of back pain and fever. Computed tomography (CT) results demonstrated aortitis with periaortic tissue thickening. DNA amplification of biopsy tissue revealed Bartonella quintana, and Bartonella serologies were subsequently noted to be positive. The patient improved with prolonged doxycycline and rifabutin treatment. This case illustrates how molecular techniques are increasingly important in diagnosing Bartonella infections.

Application of whole-genome sequencing for bacterial strain typing in molecular epidemiology.

Nosocomial infections pose a significant threat to patient health; however, the gold standard laboratory method for determining bacterial relatedness (pulsed-field gel electrophoresis [PFGE]) remains essentially unchanged 20 years after its introduction. Here, we explored bacterial whole-genome sequencing (WGS) as an alternative approach for molecular strain typing. We compared WGS to PFGE for investigating presumptive outbreaks involving three important pathogens: vancomycin-resistant Enterococcus faecium (n=19), methicillin-resistant Staphylococcus aureus (n=17), and Acinetobacter baumannii (n=15). WGS was highly reproducible (average≤0.39 differences between technical replicates), which enabled a functional, quantitative definition for determining clonality. Strain relatedness data determined by PFGE and WGS roughly correlated, but the resolution of WGS was superior (P=5.6×10(-8) to 0.016). Several discordant results were noted between the methods. A total of 28.9% of isolates which were indistinguishable by PFGE were nonclonal by WGS. For A. baumannii, a species known to undergo rapid horizontal gene transfer, 16.2% of isolate pairs considered nonidentical by PFGE were clonal by WGS. Sequencing whole bacterial genomes with single-nucleotide resolution demonstrates that PFGE is prone to false-positive and false-negative results and suggests the need for a new gold standard approach for molecular epidemiological strain typing.

Characterization of a multidrug-resistant, novel Bacteroides genomospecies.

Metronidazole- and carbapenem-resistant Bacteroides fragilis are rare in the United States. We isolated a multidrug-resistant anaerobe from the bloodstream and intraabdominal abscesses of a patient who had traveled to India. Whole-genome sequencing identified the organism as a novel Bacteroides genomospecies. Physicians should be aware of the possibility for concomitant carbapenem- and metronidazole-resistant Bacteroides infections.

Performance comparison of Illumina and ion torrent next-generation sequencing platforms for 16S rRNA-based bacterial community profiling.

High-throughput sequencing of the taxonomically informative 16S rRNA gene provides a powerful approach for exploring microbial diversity. Here we compare the performances of two common "benchtop" sequencing platforms, Illumina MiSeq and Ion Torrent Personal Genome Machine (PGM), for bacterial community profiling by 16S rRNA (V1-V2) amplicon sequencing. We benchmarked performance by using a 20-organism mock bacterial community and a collection of primary human specimens. We observed comparatively higher error rates with the Ion Torrent platform and report a pattern of premature sequence truncation specific to semiconductor sequencing. Read truncation was dependent on both the directionality of sequencing and the target species, resulting in organism-specific biases in community profiles. We found that these sequencing artifacts could be minimized by using bidirectional amplicon sequencing and an optimized flow order on the Ion Torrent platform. Results of bacterial community profiling performed on the mock community and a collection of 18 human-derived microbiological specimens were generally in good agreement for both platforms; however, in some cases, results differed significantly. Disparities could be attributed to the failure to generate full-length reads for particular organisms on the Ion Torrent platform, organism-dependent differences in sequence error rates affecting classification of certain species, or some combination of these factors. This study demonstrates the potential for differential bias in bacterial community profiles resulting from the choice of sequencing platform alone.

Whole genome sequencing indicates Corynebacterium jeikeium comprises 4 separate genomospecies and identifies a dominant genomospecies among clinical isolates.

Corynebacterium jeikeium is an opportunistic pathogen which has been noted for significant genomic diversity. The population structure within this species remains poorly understood. Here, we explore the relationships among 15 clinical isolates of C. jeikeium (reference strains K411 and ATCC 43734, and 13 primary isolates collected over a period of 7 years) through genetic, genomic, and phenotypic studies. We report a high degree of divergence among strains based on 16S ribosomal RNA (rRNA) gene and rpoB gene sequence analysis, supporting the presence of genetically distinct subgroups. Whole genome sequencing indicates genomic-level dissimilarity among subgroups, which qualify as four separate and distinct Corynebacterium species based on an average nucleotide identity (ANIb) threshold of <95%. Functional distinctions in antibiotic susceptibilities and metabolic profiles characterize two of these genomospecies, allowing their differentiation from others through routine laboratory testing. The remaining genomospecies can be classified through a biphasic approach integrating phenotypic testing and rpoB gene sequencing. The genomospecies predominantly recovered from patient specimens does not include either of the existing C. jeikeium reference strains, implying that studies of this pathogen would benefit from examination of representatives from the primary disease-causing group. The clinically dominant genomospecies also has the smallest genome size and gene repertoire, suggesting the possibility of increased virulence relative to the other genomospecies. The ability to classify isolates to one of the four C. jeikeium genomospecies in a clinical context provides diagnostic information for tailoring antimicrobial therapy and may aid in identification of species-specific disease associations.

Whole-genome sequencing for high-resolution investigation of methicillin-resistant Staphylococcus aureus epidemiology and genome plasticity.

Methicillin-resistant Staphylococcus aureus (MRSA) infections pose a major challenge in health care, yet the limited heterogeneity within this group hinders molecular investigations of related outbreaks. Pulsed-field gel electrophoresis (PFGE) has been the gold standard approach but is impractical for many clinical laboratories and is often replaced with PCR-based methods. Regardless, both approaches can prove problematic for identifying subclonal outbreaks. Here, we explore the use of whole-genome sequencing for clinical laboratory investigations of MRSA molecular epidemiology. We examine the relationships of 44 MRSA isolates collected over a period of 3 years by using whole-genome sequencing and two PCR-based methods, multilocus variable-number tandem-repeat analysis (MLVA) and spa typing. We find that MLVA offers higher resolution than spa typing, as it resolved 17 versus 12 discrete isolate groups, respectively. In contrast, whole-genome sequencing reproducibly cataloged genomic variants (131,424 different single nucleotide polymorphisms and indels across the strain collection) that uniquely identified each MRSA clone, recapitulating those groups but enabling higher-resolution phylogenetic inferences of the epidemiological relationships. Importantly, whole-genome sequencing detected a significant number of variants, thereby distinguishing between groups that were considered identical by both spa typing (minimum, 1,124 polymorphisms) and MLVA (minimum, 193 polymorphisms); this suggests that these more conventional approaches can lead to false-positive identification of outbreaks due to inappropriate grouping of genetically distinct strains. An analysis of the distribution of variants across the MRSA genome reveals 47 mutational hot spots (comprising ∼ 2.5% of the genome) that account for 23.5% of the observed polymorphisms, and the use of this selected data set successfully recapitulates most epidemiological relationships in this pathogen group.

Coinfection of Fusobacterium nucleatum and Actinomyces israelii in mastoiditis diagnosed by next-generation DNA sequencing.

Some bacterial infections involve potentially complex mixtures of species that can now be distinguished using next-generation DNA sequencing. We present a case of mastoiditis where Gram stain, culture, and molecular diagnosis were nondiagnostic or discrepant. Next-generation sequencing implicated coinfection of Fusobacterium nucleatum and Actinomyces israelii, resolving these diagnostic discrepancies.

Molecular analysis of sarcoidosis lymph nodes for microorganisms: a case-control study with clinical correlates.

INTRODUCTION: Sarcoidosis is an incurable, chronic granulomatous disease primarily involving the lungs and lymph nodes of unknown aetiology, treated with non-specific anti-inflammatory/immunosuppressive drugs. Persistently symptomatic patients worsen with a disabling, potentially fatal clinical course. To determine a possible infectious cause, we correlated in a case-control study the clinical information with the presence of bacterial DNA in sarcoidosis mediastinal lymph nodes compared with control lymph nodes resected during cancer surgery. METHODS: We retrospectively studied formalin-fixed, paraffin-embedded, mediastinal lymph nodes from 30 patients with sarcoidosis and 30 control patients with lung cancer. Nucleic acids were extracted from nodes, evaluated by ribosomal RNA PCR for bacterial 16S ribosomal DNA and the results were sequenced and compared with a bacterial sequence library. Clinical information was correlated. RESULTS: 11/30 (36.7%) of lymph nodes from patients with sarcoidosis had detectable bacterial DNA, significantly more than control patient lymph nodes (2/30, 6.7%), p=0.00516. At presentation, 19/30 (63.3%) patients with sarcoidosis were symptomatic including all patients with detectable bacterial DNA. Radiographically, there were 18 stage I and 12 stage II patients. All stage II patients were symptomatic and 75% had PCR-detectable bacteria. After a mean follow-up of 52.8±32.8 months, all patients with PCR-detectable bacteria in this series were persistently symptomatic requiring treatment. DISCUSSION: 36.6% of patients with sarcoidosis had detectable bacterial DNA on presentation, all of these patients were quite symptomatic and most were radiographically advanced stage II. These findings suggest that bacterial DNA-positive, symptomatic patients have more aggressive sarcoidosis that persists long term and might benefit from antimicrobial treatment directed against this presumed chronic granulomatous infection.

Molecular diagnosis of Actinomadura madurae infection by 16S rRNA deep sequencing.

Next-generation DNA sequencing can be used to catalog individual organisms within complex, polymicrobial specimens. Here, we utilized deep sequencing of 16S rRNA to implicate Actinomadura madurae as the cause of mycetoma in a diabetic patient when culture and conventional molecular methods were overwhelmed by overgrowth of other organisms.

Rapid 16S rRNA next-generation sequencing of polymicrobial clinical samples for diagnosis of complex bacterial infections.

Classifying individual bacterial species comprising complex, polymicrobial patient specimens remains a challenge for culture-based and molecular microbiology techniques in common clinical use. We therefore adapted practices from metagenomics research to rapidly catalog the bacterial composition of clinical specimens directly from patients, without need for prior culture. We have combined a semiconductor deep sequencing protocol that produces reads spanning 16S ribosomal RNA gene variable regions 1 and 2 (∼360 bp) with a de-noising pipeline that significantly improves the fraction of error-free sequences. The resulting sequences can be used to perform accurate genus- or species-level taxonomic assignment. We explore the microbial composition of challenging, heterogeneous clinical specimens by deep sequencing, culture-based strain typing, and Sanger sequencing of bulk PCR product. We report that deep sequencing can catalog bacterial species in mixed specimens from which usable data cannot be obtained by conventional clinical methods. Deep sequencing a collection of sputum samples from cystic fibrosis (CF) patients reveals well-described CF pathogens in specimens where they were not detected by standard clinical culture methods, especially for low-prevalence or fastidious bacteria. We also found that sputa submitted for CF diagnostic workup can be divided into a limited number of groups based on the phylogenetic composition of the airway microbiota, suggesting that metagenomic profiling may prove useful as a clinical diagnostic strategy in the future. The described method is sufficiently rapid (theoretically compatible with same-day turnaround times) and inexpensive for routine clinical use.