Identification and diversity of Actinomyces species in a clinical microbiology laboratory in the MALDI-TOF MS era.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has emerged as a reliable tool for bacterial identification. This study compared the Bruker MALDI-TOF BioTyper MS (MBT) and 16S rRNA gene sequencing for the identification of Actinomyces and Actinotignum spp. The MBT identified 68/77 (88.3%) of Actinomyces isolates to the genus-level and 44/77 (57.1%) of Actinomyces isolates to the species-level using the manufacturer's identification criteria. The MBT did not yield reliable identification for only 1/77 (1.3%) and generated no identification for 8/77 (10.4%) of the isolates. No misidentifications were found. Discordance at the species level was observed for eight isolates. Overall, the MBT demonstrated good concordance with the 16S rRNA gene sequencing with the exception of the closely related species A. naeslundii, A. viscosus and A. oris. A variety of Actinomyces spp. were isolated from orocervicofacial/dental specimens, but only a limited number of species were isolated from urine or intra-abdominal specimens. This study confirms the utility of MBT in the identification of Actinomyces spp. and describes the diversity and anatomic niche of species in human clinical specimens from various body sites.

Reducing unnecessary testing on sputum specimens from patients with cystic fibrosis: pathology stewardship in microbiology.

Chronic respiratory tract infection by Pseudomonas aeruginosa is the hallmark of established lung disease in patients with cystic fibrosis (CF). Antibiotic therapy can usually only suppress but not eradicate infection. In recent years, pulmonary infection with non-tuberculous Mycobacteria (NTM) species has also been increasing. These patients are often colonised with multiple isolates and determination of clinical significance of each isolate is difficult. The clinical value of frequent routine susceptibility testing of individual isolates is unproven, particularly since a delay in susceptibility testing is inevitable when purification of multiple cultured isolates is required to test each isolate separately. From August 2019 until December 2020 we ceased routine susceptibility testing on P. aeruginosa respiratory tract isolates from patients with CF if a previous isolate from the patient had susceptibility testing performed. We found that the proportion of P. aeruginosa isolates that had susceptibility testing performed dropped from 97% to 11% as a result of this change in laboratory process. During this time, we also ceased routine culture for acid-fast bacilli if this had been performed within the previous 6 months. We present the cost and resource savings for these changes in laboratory process and assess for clinical impact measured as hospital admissions, length of stay in hospital and mortality.

Curtobacterium flaccumfaciens septic arthritis following puncture with a Coxspur Hawthorn thorn.

Curtobacterium species are recognized plant pathogens. We report the first well-documented case of Curtobacterium human infection, a child with septic arthritis following puncture with a Coxspur Hawthorn plant thorn. The organism isolated from synovial tissue and the plant thorn was identified as Curtobacterium flaccumfaciens by 16S rRNA gene sequence analysis.

Increased diagnostic yield of routine multiplex PCR compared to clinician requested testing for detection of Trichomonas vaginalis.

Trichomonas vaginalis (TV) infection is the leading cause of non-viral sexually transmitted infection (STI) globally and is endemic in rural and remote Australia. However, current accurate prevalence data for TV in urban Australia are scarce as TV is not a notifiable infection outside of the Northern Territory (NT). This study evaluated Australian guidelines for TV testing and determined TV prevalence among patients at a large urban public hospital in Melbourne, Australia. A retrospective analysis of genitourinary samples screened for STIs by multiplex polymerase chain reaction (MPCR) between May 2017 and April 2019 was performed. A total of 7155 results (5064 females) were included in the analysis. A prevalence for TV of 1.7% (n=123) was found, which was higher than Neisseria gonorrhoeae (1.4%, n=103) but less than Chlamydia trachomatis (5%, n=358). The highest rate of TV (3%) was found in females aged 30-44 years (n = 48). Routine MPCR improved TV detection almost six-fold compared with clinician request based testing. Current targeted testing guidelines for TV were inadequate for case finding in an urban setting, and clinical request among symptomatic patients was rare. MPCR testing provides a comprehensive testing strategy for curable STI, and removes the need for clinical suspicion of TV. Implementation of MPCR for STI screening can improve TV detection in populations not normally suspected to be at risk and therefore potentially reduce disease transmission or complications associated with undiagnosed infection.

Clinical evaluation of AusDiagnostics SARS-CoV-2 multiplex tandem PCR assay.

BACKGROUND: In the context of the pandemic, the rapid emergency use authorisation of diagnostic assays for SARS-CoV-2 has meant there are few peer-reviewed published studies of clinical performance of commercial assays. AIMS: To evaluate the clinical performance of AusDiagnostics respiratory multiplex tandem PCR assay including SARS-CoV-2. METHODS: We reviewed the results following implementation of AusDiagnostics respiratory multiplex tandem PCR assay including SARS-CoV-2, and compared with an in-house RT-PCR assay at our State Reference Laboratory. RESULTS: Initial validation using AusDiagnostics coronavirus multiplex tandem PCR assay including SARS-CoV-2 demonstrated good concordance with the State Reference Laboratory. After implementing the AusDiagnostics respiratory multiplex tandem PCR assay including SARS-CoV-2, we tested 7839 samples. 127 samples in which SARS-CoV-2 was detected using the AusDiagnostics assay were referred for testing at the State Reference Laboratory, with concordant results in 118/127 (92.9%) of samples. After resolution of discrepancies, 125/127 (98.4%) of AusDiagnostics results were determined to be true positive results. Out of 7839 samples tested for SARS-CoV-2 during this period, only 2 tests (0.02%) were indeterminate results. CONCLUSION: The AusDiagnostics respiratory MT-PCR assay is a reliable assay for detection of SARS-CoV-2.

Dexamethasone Use in the Treatment of Pediatric Deep Neck Space Infections.

OBJECTIVES: Assess the outcome of Intravenous (IV) dexamethasone in the treatment of pediatric deep neck space infections (DNSI) in combination with IV antibiotics. METHODS: Retrospective chart review of pediatric patients admitted for a DNSI from March 2014 to June 2016. Patient characteristics including demographics, abscess type, antibiotic, dexamethasone, surgery, culture, and length of stay (LOS) were obtained. Patients treated with antibiotics alone versus antibiotics and dexamethasone were compared. Primary outcome measures were rate of surgical drainage and LOS. RESULTS: Overall 153 patients with DNSI were identified, including 62 lateral neck, 18 parapharyngeal, 40 peritonsillar, 32 retropharyngeal, and 1 submandibular. All patients received antibiotics. Dexamethasone was used in 35% of patients. The rate of surgical drainage in the dexamethasone and non-dexamethasone group was 36% and 53% respectively (P = .043). LOS was shorter for the dexamethasone group (2.9 days) compared to the non-dexamethasone group (3.8 days) but was non-significant, P-value-.09. The most common microorganisms cultured were MRSA (25), MSSA (11), and Streptococcus pyogenes (10). CONCLUSION: Dexamethasone use was associated with a decreased rate of surgical drainage in pediatric patients with DNSI. Further prospective study is needed to determine the role of dexamethasone in treatment.

Antineoplastic agents. 570. Isolation and structure elucidation of bacillistatins 1 and 2 from a marine Bacillus silvestris.

Two new cyclodepsipeptides designated bacillistatins 1 (1) and 2 (2) have been isolated from cultures of a sample of Bacillus silvestris that was obtained from a Pacific Ocean (southern Chile) crab. Each 12-unit cyclodepsipeptide strongly inhibited growth of a human cancer cell line panel, with GI(50)'s of 10(-4)-10(-5) microg/mL, and each compound was active against antibiotic-resistant Streptococcus pneumoniae. The structures were elucidated by a combination of X-ray diffraction and mass and 2D NMR spectroscopic analyses, together with chemical degradation.

Real-Time PCR for Diagnosing and Quantifying Co-infection by Two Globally Distributed Fungal Pathogens of Wheat.

Co-infections - invasions of a host-plant by multiple pathogen species or strains - are common, and are thought to have consequences for pathogen ecology and evolution. Despite their apparent significance, co-infections have received limited attention; in part due to lack of suitable quantitative tools for monitoring of co-infecting pathogens. Here, we report on a duplex real-time PCR assay that simultaneously distinguishes and quantifies co-infections by two globally important fungal pathogens of wheat: Pyrenophora tritici-repentis and Parastagonospora nodorum. These fungi share common characteristics and host species, creating a challenge for conventional disease diagnosis and subsequent management strategies. The assay uses uniquely assigned fluorogenic probes to quantify fungal biomass as nucleic acid equivalents. The probes provide highly specific target quantification with accurate discrimination against non-target closely related fungal species and host genes. Quantification of the fungal targets is linear over a wide range (5000-0.5 pg DNA µl-1) with high reproducibility (RSD ≤ 10%). In the presence of host DNA in the assay matrix, fungal biomass can be quantified up to a fungal to wheat DNA ratio of 1 to 200. The utility of the method was demonstrated using field samples of a cultivar sensitive to both pathogens. While visual and culture diagnosis suggested the presence of only one of the pathogen species, the assay revealed not only presence of both co-infecting pathogens (hence enabling asymptomatic detection) but also allowed quantification of relative abundances of the pathogens as a function of disease severity. Thus, the assay provides for accurate diagnosis; it is suitable for high-throughput screening of co-infections in epidemiological studies, and for exploring pathogen-pathogen interactions and dynamics, none of which would be possible with conventional approaches.

Host-Multi-Pathogen Warfare: Pathogen Interactions in Co-infected Plants.

Studies of plant-pathogen interactions have historically focused on simple models of infection involving single host-single disease systems. However, plant infections often involve multiple species and/or genotypes and exhibit complexities not captured in single host-single disease systems. Here, we review recent insights into co-infection systems focusing on the dynamics of host-multi-pathogen interactions and the implications for host susceptibility/resistance. In co-infection systems, pathogen interactions include: (i) Competition, in which competing pathogens develop physical barriers or utilize toxins to exclude competitors from resource-dense niches; (ii) Cooperation, whereby pathogens beneficially interact, by providing mutual biochemical signals essential for pathogenesis, or through functional complementation via the exchange of resources necessary for survival; (iii) Coexistence, whereby pathogens can stably coexist through niche specialization. Furthermore, hosts are also able to, actively or passively, modulate niche competition through defense responses that target at least one pathogen. Typically, however, virulent pathogens subvert host defenses to facilitate infection, and responses elicited by one pathogen may be modified in the presence of another pathogen. Evidence also exists, albeit rare, of pathogens incorporating foreign genes that broaden niche adaptation and improve virulence. Throughout this review, we draw upon examples of co-infection systems from a range of pathogen types and identify outstanding questions for future innovation in disease control strategies.

Accelerated Generation of Selfed Pure Line Plants for Gene Identification and Crop Breeding.

Production of pure lines is an important step in biological studies and breeding of many crop plants. The major types of pure lines for biological studies and breeding include doubled haploid (DH) lines, recombinant inbred lines (RILs), and near isogenic lines (NILs). DH lines can be produced through microspore and megaspore culture followed by chromosome doubling while RILs and NILs can be produced through introgressions or repeated selfing of hybrids. DH approach was developed as a quicker method than conventional method to produce pure lines. However, its drawbacks of genotype-dependency and only a single chance of recombination limited its wider application. A recently developed fast generation cycling system (FGCS) achieved similar times to those of DH for the production of selfed pure lines but is more versatile as it is much less genotype-dependent than DH technology and does not restrict recombination to a single event. The advantages and disadvantages of the technologies and their produced pure line populations for different purposes of biological research and breeding are discussed. The development of a concept of complete in vitro meiosis and mitosis system is also proposed. This could integrate with the recently developed technologies of single cell genomic sequencing and genome wide selection, leading to a complete laboratory based pre-breeding scheme.

Improved standardization and potential for shortened time to results with BD Kiestra™ total laboratory automation of early urine cultures: A prospective comparison with manual processing.

We compared the results of 505 urine specimens prospectively processed by both conventional manual processing (MP) with 16-24h incubation to BD Kiestra™ Total Laboratory Automation (TLA) system with a shortened incubation of 14h: 97% of culture results were clinically concordant. TLA processing was associated with improved standardization of time of first culture reading and total incubation time.

Impact of race on oropharyngeal squamous cell carcinoma presentation and outcomes among veterans.

BACKGROUND: Racial disparities in oropharyngeal squamous cell carcinoma (SCC) have been demonstrated and attributed to differences in human papillomavirus (HPV) status. The purpose of this study was to examine racial disparities in oropharyngeal SCC among veterans. METHODS: Retrospective review of patients with oropharyngeal SCC at a tertiary-care Veterans Affairs (VA) hospital. Adjusted Cox proportional hazards models were conducted to examine the effect of race on oropharyngeal SCC outcomes. RESULTS: Of 158 patients, 126 (79.7%) were white and 32 (20.3%) were African American. No difference in p16 tumor expression was noted between the groups. Five-year disease-free survival (DFS) was 42.6% and 55.1% for African Americans and whites, respectively (p = .372). Five-year overall survival (OS) for African Americans and whites was 54.6% and 51.8%, respectively (p = .768). On multivariate analysis, there was no significant difference in risk of recurrence or death by race. CONCLUSION: Racial disparities are largely ameliorated in patients with oropharyngeal SCC treated within the VA, there were no racial differences in p16 tumor expression, and outcomes remain poor.

Isolation of dermatophytes (and other fungi) from human nail and skin dust produced by podiatric medical treatments in Australia.

BACKGROUND: Podiatric physicians routinely use electric drills for the treatment of nail and skin conditions. The grinding process produces human nail and skin dust that is generally vacuumed into bags in the grinding unit. Many of the nails are thought to be mycotic, particularly because they are obtained from patients with symptoms of dermatophyte infections. Currently, there is limited information available on the detection of fungi from nail dust samples. Herein, we attempt to address this situation and outline some of the difficulties that pathology laboratories face in isolating and identifying dermatophytes from nail samples. METHODS: Fifty nail dust bags from podiatric medical clinics across all of the states and territories of Australia were collected and analyzed. Samples from the bags were inoculated onto primary isolation media. Fungal colonies that grew were then inoculated onto potato dextrose agar for identification using standard morphological (macroscopic and microscopic) features. RESULTS: One hundred fifty-one colonies of dermatophytes were identified from 43 of the 50 samples. In addition 471 nondermatophyte molds were isolated, along with some yeasts and bacteria. CONCLUSIONS: The most common dermatophytes isolated were from the Trichophyton mentagrophytes/interdigitale complexes. Trichophyton rubrum, Trichophyton tonsurans, Trichophyton soudanense, and Epidermophyton floccosum were also isolated. An unidentified group of dermatophytes was also present. The three most common genera of nondermatophyte molds were Aspergillus, Penicillium, and Scopulariopsis, all of which have been implicated in onychomycosis and more general disease. The presence of viable fungal pathogens in the dust could potentially pose a health problem to podiatric physicians.

Isolation of Dermatophytes (and Other Fungi) from Human Nail and Skin Dust Produced by Podiatric Medical Treatments in Australia.

Background: Podiatric physicians routinely use electric drills for the treatment of nail and skin conditions. The grinding process produces human nail and skin dust that is generally vacuumed into bags in the grinding unit. Many of the nails are thought to be mycotic, particularly because they are obtained from patients with symptoms of dermatophyte infections. Currently, there is limited information available on the detection of fungi from nail dust samples. Herein, we attempt to address this situation and outline some of the difficulties that pathology laboratories face in isolating and identifying dermatophytes from nail samples. Methods: Fifty nail dust bags from podiatric medical clinics across all of the states and territories of Australia were collected and analyzed. Samples from the bags were inoculated onto primary isolation media. Fungal colonies that grew were then inoculated onto potato dextrose agar for identification using standard morphological (macroscopic and microscopic) features. Results: One hundred fifty-one colonies of dermatophytes were identified from 43 of the 50 samples. In addition 471 nondermatophyte molds were isolated, along with some yeasts and bacteria. Conclusions: The most common dermatophytes isolated were from the Trichophyton mentagrophytes/interdigitale complexes. Trichophyton rubrum, Trichophyton tonsurans, Trichophyton soudanense, and Epidermophyton floccosum were also isolated. An unidentified group of dermatophytes was also present. The three most common genera of nondermatophyte molds were Aspergillus, Penicillium, and Scopulariopsis, all of which have been implicated in onychomycosis and more general disease. The presence of viable fungal pathogens in the dust could potentially pose a health problem to podiatric physicians.

Oropharyngeal squamous cell carcinoma in the veteran population: Association with traditional carcinogen exposure and poor clinical outcomes.

BACKGROUND: A significant fraction of oropharyngeal squamous cell carcinoma (SCC) cases is associated with traditional carcinogens; in these patients, treatment response and clinical outcomes remain poor. METHODS: We evaluated patient, tumor, and treatment characteristics for 200 veterans with oropharyngeal SCC treated at the Michael E. DeBakey Veterans Affairs Medical Center (MEDVAMC) between 2000 and 2012. RESULTS: Most patients (77%) were white and heavy smokers. Twenty-seven patients required tracheostomy and 63 required gastrostomy placement during treatment. Overall survival (OS) at 5 years was 40%. Survival was impacted by T classification, treatment intensity, completion of treatment, and p16 tumor status. Almost 30% of patients were unable to complete a treatment regimen consistent with National Comprehensive Cancer Network (NCCN) guidelines. CONCLUSION: Oropharyngeal SCC in veterans is associated with traditional carcinogens and poor clinical outcomes. Despite heavy smoking exposure, p16 tumor status significantly impacts survival. Careful consideration must be given to improving treatment paradigms for this cohort given their limited tolerance for treatment escalation.

Variation in chlorophyll content per unit leaf area in spring wheat and implications for selection in segregating material.

Reduced levels of leaf chlorophyll content per unit leaf area in crops may be of advantage in the search for higher yields. Possible reasons include better light distribution in the crop canopy and less photochemical damage to leaves absorbing more light energy than required for maximum photosynthesis. Reduced chlorophyll may also reduce the heat load at the top of canopy, reducing water requirements to cool leaves. Chloroplasts are nutrient rich and reducing their number may increase available nutrients for growth and development. To determine whether this hypothesis has any validity in spring wheat requires an understanding of genotypic differences in leaf chlorophyll content per unit area in diverse germplasm. This was measured with a SPAD 502 as SPAD units. The study was conducted in series of environments involving up to 28 genotypes, mainly spring wheat. In general, substantial and repeatable genotypic variation was observed. Consistent SPAD readings were recorded for different sampling positions on leaves, between different leaves on single plant, between different plants of the same genotype, and between different genotypes grown in the same or different environments. Plant nutrition affected SPAD units in nutrient poor environments. Wheat genotypes DBW 10 and Transfer were identified as having consistent and contrasting high and low average SPAD readings of 52 and 32 units, respectively, and a methodology to allow selection in segregating populations has been developed.

Management of obstructive sleep apnea in the indigent population: a deviation of standard of care?

Comprehensive management of patients with obstructive sleep apnea (OSA) typically is managed best via a multidisciplinary approach, involving otolaryngologists, sleep psychologists/psychiatrists, pulmonologists, neurologists, oral surgeons, and sleep trained dentists. By utilizing these resources, one could fashion a treatment individualized to the patient, giving rise to the holistic phrase of "personalized medicine." Unfortunately, in situations and environments with limited resources, the treatment options in an otolaryngologist's armamentarium are restricted--typically to continuous positive airway pressure (CPAP) versus sleep surgery. However, a recent patient encounter highlighted here shows how a hospital's reimbursement policy effectively dictated a patient's medical management to sleep surgery. This occurred although the current gold standard for the initial treatment of OSA is CPAP. Changing the course of medical/surgical management by selectively restricting funding is a cause of concern, especially when it promotes patients to choose a treatment option that is not considered the current standard of care.

Usefulness of sleep endoscopy in predicting positional obstructive sleep apnea.

OBJECTIVES: The aim of the study was to (1) evaluate whether position affects drug-induced sleep endoscopy (DISE) findings in positional and nonpositional patients and (2) determine which areas of the upper airway obstruct in different body positions. STUDY DESIGN: Prospective, case-controlled study. SETTING: Academic tertiary care center. SUBJECTS AND METHODS: Twenty-two patients with obstructive sleep apnea (OSA) were enrolled. Two groups were individually recruited to make 11 consecutive patients with positional OSA and 11 consecutive patients with nonpositional OSA. Positional OSA was defined by nonsupine 50% reduction in apnea-hypopnea index. DISE was performed with patients in both lateral and supine sleep positions. Upper airway collapse was compared between the sleep positions and between the 2 groups. RESULTS: Most patients (77%) demonstrated multilevel obstruction on DISE. Nearly all patients with positional OSA (91%) had at least a partial improvement in collapse while in the lateral sleep position. Most of the reduction in collapse involved the tongue base and epiglottis (P < .05). Sleep position did not significantly alter the upper airway morphology of patients with nonpositional OSA. Apnea-hypopnea index and body mass index were not significantly different between the 2 groups. CONCLUSIONS: Sleep position can change upper airway morphology on DISE, particularly positional OSA patients. Hypopharyngeal collapse was the primary site that improved with change in position. DISE in multiple sleep positions should be considered as part of a minimally invasive approach to surgical therapy of OSA.

Association between metformin use and improved survival in patients with laryngeal squamous cell carcinoma.

BACKGROUND: Metformin use has been linked to improved cancer outcomes. The purpose of this study was to determine the impact of metformin on the survival of patients with laryngeal cancer. METHODS: We retrospectively reviewed 205 patients with a diagnosis of laryngeal squamous cell carcinoma (SCC). RESULTS: Patients taking metformin presented with more early-stage tumors (T1 and T2) and less regional metastasis (N0; 81% metformin+ vs 50% metformin-) compared to patients with diabetes not taking metformin. At last follow-up, 76% of patients taking metformin were alive, compared to 41% for diabetics not on metformin and 51% for nondiabetics. Metformin users demonstrated increased disease-free survival (DFS) and overall survival (OS) compared with nonmetformin users and nondiabetics. CONCLUSION: Patients with diabetes taking metformin present with earlier stage disease and have more favorable clinical outcomes when compared to their diabetic counterparts and nondiabetics. Combined with previously published retrospective studies, our results suggest that prospective clinical trials evaluating the role of metformin in head and neck cancer are warranted.