Machine Learning Analysis Using RNA Sequencing to Distinguish Neuromyelitis Optica from Multiple Sclerosis and Identify Therapeutic Candidates.

This study aims to identify RNA biomarkers distinguishing neuromyelitis optica (NMO) from relapsing-remitting multiple sclerosis (RRMS) and explore potential therapeutic applications leveraging machine learning (ML). An ensemble approach was developed using differential gene expression analysis and competitive ML methods, interrogating total RNA-sequencing data sets from peripheral whole blood of treatment-naïve patients with RRMS and NMO and healthy individuals. Pathway analysis of candidate biomarkers informed the biological context of disease, transcription factor activity, and small-molecule therapeutic potential. ML models differentiated between patients with NMO and RRMS, with the performance of certain models exceeding 90% accuracy. RNA biomarkers driving model performance were associated with ribosomal dysfunction and viral infection. Regulatory networks of kinases and transcription factors identified biological associations and identified potential therapeutic targets. Small-molecule candidates capable of reversing perturbed gene expression were uncovered. Mitoxantrone and vorinostat-two identified small molecules with previously reported use in patients with NMO and experimental autoimmune encephalomyelitis-reinforced discovered expression signatures and highlighted the potential to identify new therapeutic candidates. Putative RNA biomarkers were identified that accurately distinguish NMO from RRMS and healthy individuals. The application of multivariate approaches in analysis of RNA-sequencing data further enhances the discovery of unique RNA biomarkers, accelerating the development of new methods for disease detection, monitoring, and therapeutics. Integrating biological understanding further enhances detection of disease-specific signatures and possible therapeutic targets.

Evolution of Specimen Self-Collection in the COVID-19 Era: Implications for Population Health Management of Infectious Disease.

Laboratory testing is an important component in the diagnosis of respiratory tract infections such as with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). However, specimen collection not only risks exposure of health care workers and other patients to infection, but also necessitates use of personal protective equipment that may be in short supply during periods of heightened disease activity. Self-collection of nasal or oropharyngeal swabs offers an alternative to address these drawbacks. Although studies in the past decade have demonstrated the utility of this approach for respiratory infections, it has not been widely adopted in routine clinical practice. The rapid spread of coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, has focused attention on the need for safe, convenient, timely, and scalable methods for collecting upper respiratory specimens for testing. The goals of this article are to highlight the literature regarding self-collected nasal or oropharyngeal specimens for respiratory pathogen testing; discuss the role of self-collection in helping prevent the spread of the COVID-19 disease from infected patients and facilitating a shift toward "virtual" medicine or telemedicine; and describe the current and future state of self-collection for infectious agents, and the impacts these approaches can have on population health management and disease diagnosis and prevention.

Detection of high-risk human papillomavirus infection in tonsillar specimens using 2 commercially available assays.

THE OBJECTIVE OF THE STUDY IS TO DETERMINE THE PREVALENCE OF HIGH-RISK HUMAN PAPILLOMAVIRUS (HRHPV) INFECTION IN TONSILLAR SWABS AND TISSUE: Patients undergoing tonsillectomy for nonmalignant causes were enrolled. A flocked swab and fresh tissue were collected from the left and right tonsil of each patient. Specimens were tested for hrHPV DNA using the Roche cobas test and for the presence of E6/E7 messenger RNA using the Hologic Aptima hrHPV test. Of the 193 patients enrolled, 129 were in the pediatric group (ages 1-12years; median, 5years), and 64 were in the adult group (ages 13-55; median, 22years). All swab and tissue specimens were negative for hrHPV by both methods. Positive, negative, and internal controls performed as expected. We found a 0% rate of infection indicating that detectable hrHPV infection in tonsillar tissue appears to be uncommon in the children and adults in the population sampled.

PCR-electrospray ionization mass spectrometry for direct detection of pathogens and antimicrobial resistance from heart valves in patients with infective endocarditis.

Microbiological diagnosis is pivotal to the appropriate management and treatment of infective endocarditis. We evaluated PCR-electrospray ionization mass spectrometry (PCR/ESI-MS) for bacterial and candidal detection using 83 formalin-fixed paraffin-embedded heart valves from subjects with endocarditis who had positive valve and/or blood cultures, 63 of whom had positive valvular Gram stains. PCR/ESI-MS yielded 55% positivity with concordant microbiology at the genus/species or organism group level (e.g., viridans group streptococci), 11% positivity with discordant microbiology, and 34% with no detection. PCR/ESI-MS detected all antimicrobial resistance encoded by mecA or vanA/B and identified a case of Tropheryma whipplei endocarditis not previously recognized.

Effectiveness of patient-collected swabs for influenza testing.

OBJECTIVE: To compare the effectiveness of self-collected and health care worker (HCW)-collected nasal swabs for detection of influenza viruses and determine the patients' preference for type of collection. PATIENTS AND METHODS: We enrolled adult patients presenting with influenzalike illness to the Emergency Department at Mayo Clinic, Rochester, Minnesota, from January 28, 2011, through April 30, 2011. Patients self-collected a midturbinate nasal flocked swab from their right nostril following written instructions. A second swab was then collected by an HCW from the left nostril. Swabs were tested for influenza A and B viruses by real-time reverse transcription-polymerase chain reaction, and percent concordance between collection methods was determined. RESULTS: Of the 72 paired specimens analyzed, 25 were positive for influenza A or B RNA by at least one of the collection methods (34.7% positivity rate). When the 14 patients who had prior health care training were excluded, the qualitative agreement between collection methods was 94.8% (55 of 58). Two of the 58 specimens (3.4%) from patients without health care training were positive only by HCW collection, and 1 of 58 (1.7%) was positive only by patient self-collection. A total of 53.4% of patients (31 of 58) preferred the self-collection method over the HCW collection, and 25.9% (15 of 58) had no preference. CONCLUSION: Self-collected midturbinate nasal swabs provide a reliable alternative to HCW collection for influenza A and B virus real-time reverse transcription-polymerase chain reaction.

Evaluation of the Bruker Biotyper and Vitek MS matrix-assisted laser desorption ionization-time of flight mass spectrometry systems for identification of nonfermenting gram-negative bacilli isolated from cultures from cystic fibrosis patients.

The Bruker Biotyper and Vitek MS matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) instruments were evaluated for the identification of nonfermenting gram-negative bacilli (NFGNB) by a blinded comparison to conventional biochemical or molecular methods. Two hundred NFGNB that were recovered from cultures from cystic fibrosis patients in the University of Iowa Health Care (UIHC) Microbiology Laboratory between 1 January 2006 and 31 October 2010 were sent to Mayo Clinic for analysis with the Bruker Biotyper (software version 3.0) and to bioMérieux for testing with Vitek MS (SARAMIS database version 3.62). If two attempts at direct colony testing failed to provide an acceptable MALDI-TOF identification, an extraction procedure was performed. The MS identifications from both of these systems were provided to UIHC for comparison to the biochemical or molecular identification that had been reported in the patient record. Isolates with discordant results were analyzed by 16S rRNA gene sequencing at UIHC. After discrepancy testing, the Bruker Biotyper result agreed with the biochemical or molecular method, with 72.5% of isolates to the species level, 5.5% to the complex level, and 19% to the genus level (3% not identified). The level of agreement for Vitek MS was 80% species, 3.5% complex, 6% genus, and 3.5% family (7% not identified). Both MS systems provided rapid (≤3 min per isolate) and reliable identifications. The agreement of combined species/complex/genus-level identification with the reference method was higher for the Bruker Biotyper (97% versus 89.5%, P = 0.004) but required an extraction step more often. Species-level agreement with the reference method was similar for both MS systems (72.5% and 80%, P = 0.099).

Optimized pathogen detection with 30- compared to 20-milliliter blood culture draws.

Using data from 23,313 patients, we assessed whether two blood culture sets of three bottles per set would detect more pathogens than two sets of two bottles per set and achieve similar sensitivity to collecting three sets of two bottles per set. We also compared the yield of aerobic and anaerobic bottles. Thirty milliliters of blood was distributed to one anaerobic and two aerobic bottles. Among 26,855 collections of ≥ 60 ml within 30 min, 1,379 (5.1%) were positive for a pathogen not requiring detection in more than one set to be considered a pathogen, with 72 additional distinct pathogens detected using two 30-ml compared to two 20-ml sets of one aerobic and one anaerobic bottle (increased yield, 7.9%; 95% confidence interval [CI], 6.2 to 9.8%). For conditional pathogens requiring detection in at least two positive blood cultures for classification as pathogens (i.e., otherwise classified as contaminants), there were 162 positive detections with two 30-ml sets, of which 16 would not have been detected by two 20-ml sets (increased yield, 11.0% [95% CI, 6.4 to 17.2%]). Among 134 subjects who had three sets of 30 ml each within a 30-min interval, there was complete concordance between 60 ml of blood drawn in the first two sets of 30 ml and three 20-ml sets (P = 1.0). One aerobic bottle plus one anaerobic bottle yielded more pathogens than two aerobic bottles for organisms requiring a single (P < 0.001) and two (P = 0.04) positive sets to be defined as pathogens. In conclusion, we showed that collection of two aerobic and one anaerobic blood culture bottles per set results in improved yield compared to two bottles per set. We also confirmed that an anaerobic bottle should be included in blood culture sets.

Effect of carbon dioxide on broth microdilution susceptibility testing of Brucella spp.

Since some strains of Brucella species may require carbon dioxide for growth, a multilaboratory study was conducted to compare broth microdilution susceptibility results using ambient air (AA) and 5% CO2 incubation conditions. Six antimicrobial agents were tested against 39 Brucella isolates. Aminoglycoside MICs tended to be 1 log2 dilution higher in CO2 than in AA; tetracycline-class MICs to be 1 log2 dilution lower in CO2.

Cephalosporin MIC distribution of extended-spectrum-{beta}-lactamase- and pAmpC-producing Escherichia coli and Klebsiella species.

The acquisition of beta-lactamases in members of the Enterobacteriaceae family poses a challenge to antimicrobial susceptibility testing in the clinical laboratory. We correlated the distribution of the MICs for Klebsiella spp. and Escherichia coli with the presence of extended-spectrum beta-lactamase (ESBL) and plasmid-mediated AmpC beta-lactamase (pAmpC) genes. A total of 264 isolates were subjected to cefazolin, ceftriaxone, cefotaxime, ceftazidime, cefepime, and aztreonam agar dilution MIC determination; ESBL screening and confirmatory testing by the methods of the Clinical and Laboratory Standards Institute (CLSI); and for isolates for which the MICs of extended-spectrum cephalosporins were > or =1 microg/ml or the MICs of cefpodoxime were >4 microg/ml, PCR amplification and sequencing of the ESBL and pAmpC genes. PCR was positive for 73/81 isolates (45 isolates with an ESBL gene alone, 24 isolates with a pAmpC gene alone, with 4 isolates with both genes). Compared to PCR, confirmatory testing by the CLSI method yielded a sensitivity and a specificity of 98.0 and 96.3%, respectively; there were six false-positive results and one false-negative result. No distinction in the MIC distribution was apparent between isolates with the ESBL gene and isolates with the pAmpC gene. A substantial percentage of the isolates with PCR-confirmed ESBL and/or pAmpC genes fell within the current CLSI susceptible category. For a ceftazidime, ceftriaxone, or cefotaxime MIC of > or =2 microg/ml, a dichotomy existed between isolates with and without ESBL and pAmpC genes in most cases. This suggests that the presence of the ESBL and the pAmpC enzymes may yield similar MICs of extended-spectrum cephalosporins, many of which fall within the current nonresistant categories. Lowering of the current CLSI breakpoints for cephalosporins appears to be warranted.

Real-time PCR vs standard culture detection of group A beta-hemolytic streptococci at various anatomic sites in tonsillectomy patients.

OBJECTIVE: To compare rates of group A beta-hemolytic streptococci (GABHS) detection by real-time polymerase chain reaction (rtPCR) and standard culture (SCx) at different anatomic sites to determine whether a more patient-friendly site (eg, retromolar trigone or gingivobuccal sulcus) would yield results similar to the tonsillar surface. Real-time polymerase chain reaction can detect GABHS at rates equal to SCx, and results require only a few hours. DESIGN: Prospective study. SETTING: Tertiary care setting. PATIENTS: The study population comprised 130 patients undergoing tonsillectomy or adenotonsillectomy. INTERVENTION: At tonsillectomy, swabs were taken of pharyngeal tonsil surface, pharyngeal tonsillar core, inferior gingivobuccal sulcus, and retromolar trigone. Tissue samples were taken from tonsil core and adenoid. All comparisons between methods and sites were made using the McNemar test for comparing correlated proportions. All calculated P values were 2-sided. MAIN OUTCOME MEASURE: Detection of GABHS by rtPCR and SCx. RESULTS: In 41 cases (32%), GABHS was detected at 1 or more sampled sites, and 29 of those positive were detected on the tonsil surface-SCx and rtPCR results were both positive in 28 (97%). Of these 29 cases, results from the gingivobuccal site were positive by both rtPCR and SCx in 4 (14%), rtPCR only in 3 (10%), and SCx only in 3 (10%). Of the 7 tonsil surface-positive cases with retromolar trigone swabs, results were positive by rtPCR only in 1 (14%) and SCx only in 2 (29%). CONCLUSION: Whether rtPCR or SCx is used, swabs of gingivobuccal sulcus and retromolar trigone do not accurately reflect GABHS populations on the tonsil surface.

Bivalvular Bartonella henselae prosthetic valve endocarditis.

Prosthetic valve endocarditis is an uncommon manifestation of infection with Bartonella species. Herein, we report a case of Bartonella henselae endocarditis involving prosthetic mitral and aortic valves. The patient had a favorable outcome with combined medical and surgical therapy. Concomitant crescentic glomerulonephritis led to an initial mistaken diagnosis of Wegener's granulomatosis.

Quantitative real-time polymerase chain reaction for evaluating DNAemia due to cytomegalovirus, Epstein-Barr virus, and BK virus in solid-organ transplant recipients.

Testing for cytomegalovirus-, Epstein-Barr virus-, and BK virus-specific gene targets in specimens from solid-organ transplant recipients for DNA by quantitative real-time polymerase chain reaction has been implemented in many diagnostic facilities. This technology provides rapid, accurate, and reproducible results for early detection, monitoring, and medical management of patients with these infections. Because these assays are becoming commonly used in clinical practice, the technical variables associated with specimen processing (e.g., nucleic acid extraction, gene target, and result reporting), amplification, and unique patient characteristics (e.g., age, sex, underlying diseases, immune status, and immunosuppressive regimens received) are factors that may influence the understanding and interpretation of test results. We emphasize the need for standardization of existing variables through parallel comparative and proficiency testing, uniform units for expressing results, to provide for clinical correlation with the results of these molecular assays.

Sonication of removed hip and knee prostheses for diagnosis of infection.

BACKGROUND: Culturing of samples of periprosthetic tissue is the standard method used for the microbiologic diagnosis of prosthetic-joint infection, but this method is neither sensitive nor specific. In prosthetic-joint infection, microorganisms are typically present in a biofilm on the surface of the prosthesis. We hypothesized that culturing of samples obtained from the prosthesis would improve the microbiologic diagnosis of prosthetic-joint infection. METHODS: We performed a prospective trial comparing culture of samples obtained by sonication of explanted hip and knee prostheses to dislodge adherent bacteria from the prosthesis with conventional culture of periprosthetic tissue for the microbiologic diagnosis of prosthetic-joint infection among patients undergoing hip or knee revision or resection arthroplasty. RESULTS: We studied 331 patients with total knee prostheses (207 patients) or hip prostheses (124 patients); 252 patients had aseptic failure, and 79 had prosthetic-joint infection. With the use of standardized nonmicrobiologic criteria to define prosthetic-joint infection, the sensitivities of periprosthetic-tissue and sonicate-fluid cultures were 60.8% and 78.5% (P<0.001), respectively, and the specificities were 99.2% and 98.8%, respectively. Fourteen cases of prosthetic-joint infection were detected by sonicate-fluid culture but not by prosthetic-tissue culture. In patients receiving antimicrobial therapy within 14 days before surgery, the sensitivities of periprosthetic tissue and sonicate-fluid culture were 45.0% and 75.0% (P<0.001), respectively. CONCLUSIONS: In this study, culture of samples obtained by sonication of prostheses was more sensitive than conventional periprosthetic-tissue culture for the microbiologic diagnosis of prosthetic hip and knee infection, especially in patients who had received antimicrobial therapy within 14 days before surgery.

Age- and sex-associated trends in bloodstream infection: a population-based study in Olmsted County, Minnesota.

BACKGROUND: Despite increasing concerns about antimicrobial resistance and emerging pathogens among blood culture isolates, contemporary population-based data on the age- and sex-specific incidence of bloodstream infections (BSIs) are limited. METHODS: Retrospective, population-based, cohort study of all residents of Olmsted County, Minnesota, with a BSI between January 1, 2003, and December 31, 2005. The medical record linkage system of the Rochester Epidemiology Project and microbiology records were used to identify incident cases. RESULTS: A total of 1051 unique patients with positive blood culture results were identified; 401 (38.2%) were classified as contaminated. Of 650 patients with cultures deemed clinically relevant, the mean +/- SD age was 63.1 +/- 23.1 years, and 52.5% were male. The most common organisms identified were Escherichia coli (in 163 patients with BSIs [25.1%]) and Staphylococcus aureus (in 108 patients with BSIs [16.6%]). Nosocomial BSIs were more common in males than females (23.8% vs 13.9%; P = .002). The age-adjusted incidence rate of BSI was 156 per 100 000 person-years for females and 237 per 100 000 person-years for males (P<.001), with an age- and sex-adjusted rate of 189 per 100 000 person-years. Rates of BSI due to gram-positive cocci were 64 per 100 000 person-years for females and 133 per 100 000 person-years for males (P<.001); gram-negative bacillus BSI rates (85/100 000 person-years for females and 79/100 000 person-years for males) were not significantly different between sexes (P = .79). The rate of S aureus BSI was 23 per 100 000 person-years for females and 46 per 100 000 person-years for males (P = .005). CONCLUSIONS: There are significant differences in the age and sex distribution of organisms among patients with BSIs. The incidence of BSI increases sharply with increasing age and is significantly higher in males, mainly because of nosocomial organisms, including S aureus.

Unusual aerobic and anaerobic bacteria associated with prosthetic joint infections.

The association of certain microorganisms, such as Staphylococcus epidermidis, Staphylococcus aureus, and beta-hemolytic streptococci, with prosthetic joint infection (PJI) has been recognized for many years. To our knowledge, a systematic review of the presentation and management of less commonly encountered species of coagulase-negative staphylococci, nutritional-variant streptococci, aerobic non-spore and spore forming Gram-positive or anaerobic bacteria is not available. We therefore sought to provide a comprehensive literature review of PJI due to these microorganisms that will provide a valuable and quick reference for clinicians caring for these patients. We conducted a Medline search of all case reports and case series of PJI due to unusual aerobic and anaerobic bacteria. The presentation, surgical, and medical management strategies were reviewed. Appropriate medical and surgical management of such infections is complex and evolving as newer diagnostic tests, surgical techniques and antimicrobials become available. Management of patients with these infections requires close collaboration between the orthopaedic surgeon, infectious disease specialist and microbiology laboratory.

Fungi, mycobacteria, zoonotic and other organisms in prosthetic joint infection.

A systematic review of the presentation and management of prosthetic joint infection (PJI) due to zoonotic microorganisms, fungi, mycobacteria and other unusual microorganisms is not available. We therefore sought to provide a resource for the practicing orthopaedic surgeon which will serve as a guide for making appropriate decisions in managing such rare, but potentially problematic conditions. We conducted a Medline search of all case reports of PJI due to these unusual microorganisms. Our review of the current literature showed the diagnosis of PJI due to zoonotic microorganisms, fungi, mycobacteria and other unusual microorganisms typically necessitates specialized diagnostic tests. Maintaining a high index of suspicion in diagnosis of such unusual microorganisms and requesting the appropriate laboratory tests at the time of surgical débridement is crucial for determining the microbiologic etiology of these infections. The appropriate medical and surgical management of such infections is complex and often requires the use of special antimicrobials with which the orthopaedist may not be familiar. Collaboration with an infectious disease specialist is advisable when treating these infections.

Bronchoscopy in ventilator-associated pneumonia: agreement of calibrated loop and serial dilution.

RATIONALE: Although the serial dilution technique for quantitative culture of bronchoalveolar fluid is considered to be the gold standard for the diagnosis of ventilator-associated pneumonia, it is more labor intensive than the calibrated loop technique. OBJECTIVE: We sought to determine the agreement between the calibrated loop and serial dilution techniques in the diagnosis of ventilator-associated pneumonia. METHODS: We prospectively measured bacterial colony counts by the serial dilution and calibrated loop techniques in 121 bronchoalveolar lavage samples of 104 patients with suspected ventilator-associated pneumonia. MEASUREMENTS AND MAIN RESULTS: At the time of bronchoscopy, patients had received mechanical ventilation for a median of 8 d. Patients were receiving antibiotics when 90 of the 121 (74.4%) bronchoalveolar samples were obtained. The colony counts of 13 bacterial isolates were too numerous to count by the calibrated loop technique; by serial dilution technique, their counts ranged from 4.70 to 6.74 log10 cfu/ml. Fifty other bacteria had paired colony counts measured by each of the two techniques: the bias (95% confidence interval) between the two techniques was -0.380 (-0.665 to -0.095) log10 cfu/ml, with precision of 1.002 log10 cfu/ml and 95% limits of agreement of -2.344 to 1.584 log10 cfu/ml. Using the threshold of 4 log10 cfu/ml as a criterion for the diagnosis of ventilator-associated pneumonia, there was discordance only for one bacterial organism between the two techniques. CONCLUSIONS: The calibrated loop technique can be used for the diagnosis of ventilator-associated pneumonia using bronchoalveolar lavage fluid.

Sonication of explanted prosthetic components in bags for diagnosis of prosthetic joint infection is associated with risk of contamination.

Explanted orthopedic implants from 54 patients with aseptic failure and 24 patients with prosthetic knee or hip infection were sonicated in polyethylene bags. The sensitivities of periprosthetic tissue and sonicate fluid cultures for the diagnosis of prosthetic joint infection were 54% and 75%, whereas the specificities were 98% and 87%, respectively. Sonication in bags improved bacterial recovery from the surface of orthopedic implants; however, it lacked specificity, due to bag leakage.