Comparison of Bruker Biotyper® and Vitek® MS matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry platforms for the identification of filamentous fungi.

Aims: To evaluate the performance of two matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry platforms to identify molds isolated from clinical specimens. Methods: Fifty mold isolates were analyzed on Bruker Biotyper® and Vitek® MS platforms. Two Bruker Biotyper extraction protocols were assessed alongside the US FDA-approved extraction protocol for Vitek MS. Results: The Bruker Biotyper modified NIH-developed extraction protocol correctly identified more isolates than Bruker's protocol (56 vs 33%). For species in the manufacturers' databases, Vitek MS correctly identified 85% of isolates, with 8% misidentifications. The Bruker Biotyper identified 64%, with no misidentifications. For isolates not in the databases, the Bruker Biotyper did not misidentify any, and Vitek MS misidentified 36%. Conclusion: Both the Vitek MS and Bruker Biotyper accurately identified the fungal isolates, however Vitek MS was more likely to misidentify isolates than the Bruker Biotyper.

There are two different mass spectrometry systems that can be used in the hospital laboratory to find out what kind of mold is growing from a patient sample: the Vitek^® MS and Bruker Biotyper^® systems. This study compared how well they work for mold identification and also looked at two different ways to prepare the mold for testing. The Vitek MS system identified more molds, but also made more mistakes when identifying them. The Bruker Biotyper identified fewer molds but did not make any mistakes on the identification. The Vitek MS system sometimes gets the type of mold wrong, so more tests may be needed to be sure of the result. The Bruker Biotyper is more accurate because it got all of the molds correct, but it could not identify as many.

Prevalence and predictors of urine culture contamination in primary care: A cross-sectional study.

BACKGROUND: Antimicrobial resistance is a global health threat. To slow resistance and preserve antibiotics, stewardship interventions are increasingly promoted and mandated. Urine cultures are the most common microbiological test in the outpatient setting. Contamination most likely occurs during urine collection from surrounding vaginal, perineal, and epidermal flora. Sample contamination can lead to incorrect diagnosis, unnecessary or inappropriate treatment, poor patient outcomes, and higher costs. Therefore, ensuring proper collection of urinary samples serves as a prime diagnostic stewardship target, one that international nursing societies increasingly endorse as an opportunity for nurse involvement. OBJECTIVES: Determine the prevalence, predictors, and antibiotic prescribing associated with contaminated urine cultures in primary care clinics. DESIGN: Cross-sectional study. SETTING: Two adult safety-net clinics in Houston, Texas. PARTICIPANTS: 1265 clinical encounters among 1114 primary care patients. METHODS: We reviewed charts from office visits among patients who had a urine culture ordered between November 2018 and March 2020. Patient demographics, culture results and prescription orders were captured for each visit. Culture results were defined as no growth, contaminated (i.e., mixed flora, non-uropathogens, or ≥3 bacterial species isolated), or low-count (102-105 colony forming units (CFU)/mL) or high-count (>105 CFU/mL) uropathogen-positive. We performed multinomial logistic regression to identify predictors independently associated with contaminated cultures. RESULTS: Our study evaluated 1265 cultures from 1114 patients that were primarily female (84 %), of Hispanic/Latino (74.4 %) or Black/African American (18.9 %) race/ethnicity with a mean age of 43 years. Out of 1265 urine cultures, 264 (20.9 %) had no growth, 694 (54.9 %) were contaminated, 159 (12.6 %) were low-count positive, and 148 (11.7 %) were high-count positive. Female sex, pregnancy, and obesity were associated with contaminated cultures (multinomial adjusted odds ratios: 15.89, 14.34, 1.93, respectively; 95 % confidence intervals: 10.25-24.61, 8.03-25.61, 1.32-2.81, respectively). Antibiotic prescribing was significantly higher among symptomatic patients with contaminated cultures compared to those with no growth. CONCLUSION: Urine culture contamination occurred frequently in our clinics, and obesity, female sex and pregnancy were independent risk factors for contamination. The association of pregnancy and contamination is particularly concerning as pregnant females are routinely screened and treated for asymptomatic bacteriuria in the United States. Culture contamination may obscure underlying uropathogens, leading to pyelonephritis or potential neonatal infection if untreated. Conversely, overtreatment of false positive bacteriuria could lead to adverse effects from antibiotics and increased risk for antibiotic resistance. As nurses play a prominent role in patient education, diagnostic stewardship interventions may want to utilize nurses' educational capabilities to improve urine culture collection. TWEETABLE ABSTRACT: 55 % of urine cultures collected in primary care clinics were contaminated, revealing a major opportunity for nurse-driven diagnostic stewardship interventions.

Corrigendum: Development of phage cocktails to treat E. coli catheter-associated urinary tract infection and associated biofilms.

[This corrects the article DOI: 10.3389/fmicb.2022.796132.].

Clinical Utility of the Signal-to-Cutoff Ratio of Reactive HIV Antigen/Antibody Screening Tests in Guiding Emergency Physician Management.

BACKGROUND: The signal-to-cutoff (S/CO) ratio of the HIV antigen/antibody test may help immediately to differentiate true-positive results from false-positive results, which may be particularly useful in time-sensitive circumstances, such as when providing emergency department (ED) care. SETTING: Seven US EDs with HIV screening programs using HIV antigen/antibody assays. METHODS: This cross-sectional study of existing data correlated S/CO ratios with confirmed HIV status. Test characteristics at predetermined S/CO ratios and the S/CO ratio with the best performance by receiver operator characteristic (ROC) curve were calculated. RESULTS: Of 1035 patients with a reactive HIV antigen/antibody test, 232 (22.4%) were confirmed HIV-negative and 803 (77.6%) were confirmed HIV-positive. Of the 803 patients, 713 (88.8%) experienced chronic infections and 90 (11.2%) experienced acute infections. S/CO ratios were greater for HIV-positive (median 539.2) than for HIV-negative patients (median 1.93) (P < 0.001) and lower for acute infection (median 22.8) than for chronic infection (median 605.7) (P < 0.001). All patients with an S/CO ratio < 1.58 (n = 93) were HIV-negative (NPV 100%), and nearly all with an S/CO ≥ 20.7 (n = 760) (optimal level by ROC analysis) were HIV-positive (PPV 98.6%). Of patients with S/CO values between 1.58 and 20.7 (n = 182), 29.7% were HIV-positive. CONCLUSIONS: The S/CO ratio may be used in real time to classify most ED patients as almost certain to be either HIV-positive or HIV-negative long before nucleic acid confirmatory testing is available. When combined with clinical judgment, this could guide preliminary result disclosure and management.

Identifying Causative Microorganisms in Left Ventricular Assist Device Infections as a Guide for Developing Bacteriophage Therapy.

BACKGROUND: As more left ventricular-assist devices (LVADs) are implanted, multidrug-resistant LVAD infections are becoming increasingly common, partly due to bacterial biofilm production. To aid in developing bacteriophage therapy for LVAD infections, we have identified the most common bacterial pathogens that cause LVAD driveline infections (DLIs) in our heart transplant referral center. MATERIALS AND METHODS: We studied a retrospective cohort of patients who received LVADs from November 2003 to August 2017 to identify the common causative organisms of LVAD infection. We also studied a prospective cohort of patients diagnosed with DLIs from October 2018 to May 2019 to collect bacterial strains from DLIs for developing bacteriophages to lyse causative pathogens. LVAD infections were classified as DLI, bacteremia, and pump/device infections in the retrospective cohort. RESULTS: In the retrospective cohort of 582 patients, 186 (32.0%) developed an LVAD infection, with 372 microbial isolates identified. In the prospective cohort, 96 bacterial strains were isolated from 54 DLIs. The microorganisms causing DLIs were similar in the two cohorts; the most common isolate was Staphylococcus aureus. We identified 6 prospective S. aureus strains capable of biofilm formation. We developed 3 bacteriophages that were able to lyse 5 of 6 of the biofilm-forming S. aureus strains. CONCLUSIONS: Similar pathogens caused LVAD DLIs in our retrospective and prospective cohorts, indicating our bacterial strain bank will be representative of future DLIs. Our banked bacterial strains will be useful in developing phage cocktails that can lyse ≥80% of the bacteria causing LVAD infections at our institution.

Rapidly Fatal Postlaparoscopic Liver Infection from the Rarely Isolated Species Clostridium butyricum.

We report a case of a rapidly fatal postlaparoscopic cholecystectomy liver infection from the rarely isolated species Clostridium butyricum. Liver examination at autopsy showed cystic spaces, necrosis, and spore-forming Gram-positive rods. 16sRNA gene sequencing of the cystic liver tissue identified the organism as C. butyricum.

Misidentification of Scedosporium boydii Infection as Aspergillosis in a Patient with Chronic Renal Failure.

Aspergillosis is a commonly diagnosed fungal infection. Histopathologic examination alone can have diagnostic pitfalls due to the overlapping of fungal morphology. We report a case of Scedosporium boydii infection initially misdiagnosed as aspergillosis. The patient presented to the hospital with shortness of breath and chest and abdominal pain. Laboratory tests revealed leukocytosis and elevated serum liver enzymes, myoglobin and lipase. He died of hypotensive shock and brain abscesses despite antibiotic treatment. Autopsy revealed invasive fungal infection in the heart, thyroid, and brain with presence of 45-degree angled, branching hyphae. The initial diagnosis of aspergillosis was made; however, further molecular studies identified the organism as S. boydii. This report reveals the potential pitfalls of morphologic diagnosis alone; and the necessity of other testing modalities to render an accurate diagnosis which is crucial for appropriate.

Visualization of microbes by 16S in situ hybridization in term and preterm placentas without intraamniotic infection.

BACKGROUND: Numerous reports have documented bacteria in the placental membranes and basal plate decidua in the absence of immunopathology using histologic techniques. Similarly, independent metagenomic characterizations have identified an altered taxonomic makeup in association with spontaneous preterm birth. Here we sought to corroborate these findings by localizing presumptive intact bacteria using molecular histology within the placental microanatomy. OBJECTIVE: Here we examined for microbes in term and preterm gestations using a signal-amplified 16S universal in situ hybridization probe set for bacterial rRNA, alongside traditional histologic methods of Warthin-Starry and Gram stains, as well as clinical culture methodologies. We further sought to differentiate accompanying 16S gene sequencing taxonomic profiles from germ-free (gnotobiotic) mouse and extraction and amplicon contamination controls. STUDY DESIGN: Placentas were collected from a total of 53 subjects, composed of term labored (n = 4) and unlabored cesarean deliveries (n = 22) and preterm vaginal (n = 18) and cesarean deliveries (n = 8); a placenta from a single subject with clinical and histologic evident choriomanionitis was employed as a positive control (n = 1). The preterm cohort included spontaneous preterm birth with (n = 6) and without (n = 10) preterm premature rupture of membranes, as well as medically indicated preterm births (n = 10). Placental microbes were visualized using an in situ hybridization probe set designed against highly conserved regions of the bacterial 16S ribosome, which produces an amplified stable signal using branched DNA probes. Extracted bacterial nucleic acids from these same samples were subjected to 16S rRNA metagenomic sequencing (Illumina, V4) for course taxonomic analysis, alongside environmental and kit contaminant controls. A subset of unlabored, cesarean-delivered term pregnancies were also assessed with clinical culture for readily cultivatable pathogenic microbes. RESULTS: Molecular in situ hybridization of bacterial rRNA enabled visualization and localization of low-abundance microbes after systematic high-power scanning. Despite the absence of clinical or histologic chorioamnionitis in 52 of 53 subjects, instances of 16S rRNA signal were confidently observed in 13 of 16 spontaneous preterm birth placentas, which was not significantly different from term unlabored cesarean specimens (18 of 22; P > .05). 16S rRNA signal was largely localized to the villous parenchyma and/or syncytiotrophoblast, and less commonly the chorion and the maternal intervillous space. In all term and unlabored cesarean deliveries, visualization of evident placental microbes by in situ hybridization occurred in the absence of clinical or histologic detection using conventional clinical cultivation, hematoxylin-eosin, and Gram staining. In 1 subject, appreciable villous bacteria localized to an infarction, where 16S microbial detection was confirmed by Warthin-Starry stain. In all instances, parallel sample principle coordinate analysis using Bray-Cutis distances of 16S rRNA gene sequencing data demonstrated consistent taxonomic distinction from all negative or potential contamination controls (P = .024, PERMANOVA). Classification from contaminant filtered data identified a distinct taxonomic makeup among term and preterm cohorts when compared with contaminant controls (false discovery rate <0.05). CONCLUSION: Presumptively intact placental microbes are visualized as low-abundance, low-biomass and sparse populations within the placenta regardless of gestational age and mode of delivery. Their taxonomic makeup is distinct from contamination controls. These findings further support several previously published findings, including our own, which have used metagenomics to characterize low-abundance and low-biomass microbial communities in the placenta.

Economic health care costs of blood culture contamination: A systematic review.

BACKGROUND: Blood culture contamination with gram-positive organisms is a common occurrence in patients suspected of bloodstream infections, especially in emergency departments. Although numerous research studies have investigated the cost implications of blood culture contamination, a contemporary systematic review of the literature has not been performed. The aim of this project was to perform a systematic review of the published literature on the economic costs of blood culture contamination. METHODS: PubMed was searched (January 1, 1978, to July 15, 2018) using the search terms "blood culture contamination" or "false-positive blood cultures." Articles were title searched and abstracts were reviewed for eligible articles that reported immediate or downstream economic costs of blood culture contamination. RESULTS AND DISCUSSION: The PubMed search identified 151 relevant articles by title search, with 49 articles included after abstract review. From the studies included, overall blood culture contamination rates ranged from 0.9%-41%. Up to 59% of patients received unnecessary treatment with parenteral vancomycin as a result of blood culture contamination, resulting in increased pharmacy charges between $210 and $12,611 per patient. Increases in total laboratory charges between $2,397 and $11,152 per patient were reported. Attributable hospital length of stay increases due to blood culture contamination ranged from 1-22 days. CONCLUSIONS: This systematic review of the literature identified several areas of health care expenditure associated with blood culture contamination. Interventions to reduce the risk of blood culture contamination would avoid downstream economic costs.

Clinical Importance of Placental Testing among Suspected Cases of Congenital Zika Syndrome.

Contemporaneous Zika virus (ZIKV) strains can cause congenital Zika syndrome (CZS). Current ZIKV clinical laboratory testing strategies are limited and include IgM serology (which may wane 12 weeks after initial exposure) and nucleic acid testing (NAT) of maternal serum, urine, and placenta for (+) strand ZIKV RNA (which is often transient). The objectives of this study were to determine if use of additional molecular tools, such as quantitative PCR and microscopy, would add to the diagnostic value of current standard placental ZIKV testing in cases with maternal endemic exposure and indeterminate testing. ZIKV RNA was quantified from dissected sections of placental villi, chorioamnion sections, and full cross-sections of umbilical cord in all cases examined. Quantitation with high-resolution automated electrophoresis determined relative amounts of precisely verified ZIKV (74-nt amplicons). In order to localize and visualize stable and actively replicating placental ZIKV in situ, labeling of flaviviridae glycoprotein, RNA ISH against both (+) and (⁻) ZIKV-specific ssRNA strands, and independent histologic examination for significant pathologic changes were employed. We demonstrate that the use of these molecular tools added to the diagnostic value of placental ZIKV testing among suspected cases of congenital Zika syndrome with poorly ascribed maternal endemic exposure.

Correction: Molecular Subtypes in Head and Neck Cancer Exhibit Distinct Patterns of Chromosomal Gain and Loss of Canonical Cancer Genes.

[This corrects the article DOI: 10.1371/journal.pone.0056823.].

An in-house assay for BK polyomavirus quantification using the Abbott m2000 RealTime system.

BK polyomavirus (BKPyV) quantification is useful for monitoring renal transplant patient response to therapy. The Abbott m2000 RealTime System employed by some clinical laboratories to perform US Food and Drug Administration-approved assays can also be used to develop in-house assays such as the one presented here. This study aimed to validate an in-house quantitative real-time PCR assay targeting the BKPyV major capsid VP1 gene for assessment of viral load using the Abbott m2000 RealTime System. BKPyV load was measured in 95 urine and plasma samples previously tested for BKPyV by one of three laboratories (46 BKPyV-positive samples consisting of 35 plasma and 11 urine samples; 49 samples negative for BKPyV consisting of 47 plasma and two urine samples). Two additional plasma specimens from the College of American Pathologists proficiency testing survey were also analysed. Precision studies were performed by diluting a high-viral-titre patient sample into BKPyV-negative pooled plasma to create high-positive (6.16 log10 copies ml(-1)) and low-positive (3.16 log10 copies ml(-1)) samples. For precision studies of inter-assay variability, a high-positive (7.0 log10 copies ml(-1)) and a low-positive (3.0 log10 copies ml(-1)) sample were measured in 20 separate runs. The assay's limit of quantification and limit of detection were 2.70 and 2.25 log10 copies ml(-1), respectively. The assay was linear from 2.70 to 9.26 log10 copies ml(-1). Of the 48 known positives, 43 were detected as positive, with three reported by the reference laboratory as values lower than the limit of detection. Two known positives at 3.27 and 3.80 log10 copies ml(-1) tested negative by the m2000 BKPyV assay. Of the 49 known negative samples, 48 were negative by the m2000 BKPyV load assay, with one sample confirmed positive by a reference laboratory. Qualitative analysis prior to discrepancy testing demonstrated a sensitivity of 89.58 % and a specificity of 97.96 %. Precision studies demonstrated inter-assay coefficients of variation of 0.63 % (high positive) and 4.38 % (low positive). Genotyping was performed on 22 patient samples, of which 21 (95.45 %) were type I and one (4.55 %) was type II. In conclusion, the m2000 BKPyV viral load assay sensitivity, specificity, linear range, precision and cost effectiveness make it an attractive methodology for clinical laboratories using the Abbott m2000 RealTime System.

Prediction of lung cancer histological types by RT-qPCR gene expression in FFPE specimens.

Lung cancer histologic diagnosis is clinically relevant because there are histology-specific treatment indications and contraindications. Histologic diagnosis can be challenging owing to tumor characteristics, and it has been shown to have less-than-ideal agreement among pathologists reviewing the same specimens. Microarray profiling studies using frozen specimens have shown that histologies exhibit different gene expression trends; however, frozen specimens are not amenable to routine clinical application. Herein, we developed a gene expression-based predictor of lung cancer histology for FFPE specimens, which are routinely available in clinical settings. Genes predictive of lung cancer histologies were derived from published cohorts that had been profiled by microarrays. Expression of these genes was measured by quantitative RT-PCR (RT-qPCR) in a cohort of patients with FFPE lung cancer. A histology expression predictor (HEP) was developed using RT-qPCR expression data for adenocarcinoma, carcinoid, small cell carcinoma, and squamous cell carcinoma. In cross-validation, the HEP exhibited mean accuracy of 84% and κ = 0.77. In separate independent validation sets, the HEP was compared with pathologist diagnoses on the same tumor block specimens, and the HEP yielded similar accuracy and precision as the pathologists. The HEP also exhibited good performance in specimens with low tumor cellularity. Therefore, RT-qPCR gene expression from FFPE specimens can be effectively used to predict lung cancer histology.

Molecular subtypes in head and neck cancer exhibit distinct patterns of chromosomal gain and loss of canonical cancer genes.

Head and neck squamous cell carcinoma (HNSCC) is a frequently fatal heterogeneous disease. Beyond the role of human papilloma virus (HPV), no validated molecular characterization of the disease has been established. Using an integrated genomic analysis and validation methodology we confirm four molecular classes of HNSCC (basal, mesenchymal, atypical, and classical) consistent with signatures established for squamous carcinoma of the lung, including deregulation of the KEAP1/NFE2L2 oxidative stress pathway, differential utilization of the lineage markers SOX2 and TP63, and preference for the oncogenes PIK3CA and EGFR. For potential clinical use the signatures are complimentary to classification by HPV infection status as well as the putative high risk marker CCND1 copy number gain. A molecular etiology for the subtypes is suggested by statistically significant chromosomal gains and losses and differential cell of origin expression patterns. Model systems representative of each of the four subtypes are also presented.

Cryptococcal retropharyngeal abscess.

Cryptococcus neoformans is an opportunistic fungal pathogen that causes a variety of diseases in immunosuppressed patients. We describe a patient with sarcoidosis and odynophagia admitted with a large retropharyngeal abscess. Aspiration showed budding yeast and culture identified C. neoformans, confirmed by D2 large subunit rRNA gene sequencing.

Triplex real-time polymerase chain reaction assay for simultaneous detection of Staphylococcus aureus and coagulase-negative staphylococci and determination of methicillin resistance directly from positive blood culture bottles.

We describe here a 1-step, triplex real-time polymerase chain reaction (PCR) assay for the detection and identification of staphylococci directly from signal-positive blood culture bottles containing Gram-positive cocci in clusters (GPCC). The triplex assay targeted and detected tuf, nuc, and mecA genes in a single tube and had a detection limit of 10(5) CFU/mL for each gene target. A total of 341 GPCC-positive blood culture bottles were collected between November 12, 2008, and August 11, 2009. Among them, 230 methicillin-resistant coagulase-negative staphylococci (CoNS), 54 methicillin-susceptible CoNS, 22 methicillin-resistant Staphylococcus aureus, 22 methicillin-susceptible S. aureus, and 13 nonstaphylococci species were identified by conventional methods. The results obtained by triplex assay were in agreement with those of conventional methods for tuf (99.7%), nuc (100.0%), and mecA (99.1%), respectively. The triplex assay was found to have sensitivities of 99.7%, 100%, and 99.2% and specificities of 100%, 100%, and 98.7%, respectively, for the tuf, nuc, and mecA gene targets. The triplex real-time PCR assay accurately detects and identifies staphylococci directly from positive blood cultures without nucleic acid extraction prior to amplification.

Molecular diagnostics of infectious diseases.

PURPOSE OF REVIEW: The purpose of this article is to review the molecular methods commonly used in medical microbiology as well as to update the clinician as to newer molecular technologies that show promise in the identification of microorganisms as well as evaluation of the presence of virulence factors and antibiotic resistance determinants. RECENT FINDINGS: Numerous molecular assays have been developed recently using a variety of technologies. Direct hybridization techniques have allowed analysis of blood culture bottles for organisms such as methicillin-resistant Staphylococcus aureus. Target amplification methods allow postamplification analysis using a variety of technologies depending on the clinical needs for the assay. Postamplification analysis includes methods such as Sanger sequencing, pyrosequencing, reverse hybridization, and Luminex analysis, which are becoming more widely utilized. In the future, whole genome sequencing, mass spectrometry, and microarray analysis may provide a wealth of information that can be used to specifically tailor the treatment of infectious diseases. SUMMARY: The implications of current trends in molecular infectious diseases are moving towards high-throughput, simple, array-type technologies that will provide a wealth of data regarding types of organisms present in a sample and the virulence factors/resistance determinants that influence the severity of disease. As a result of these developments, infectious diseases will be more accurately and effectively treated.

Growth of adenocarcinoma on routine microbiological media inoculated with fluid from a pleural effusion in an 82-year-old female.

We report the first documented case of adenocarcinoma cell growth on routine microbiological media. Pleural fluid culture from an 82-year-old female showed colonies with fried egg appearance on routine microbiological media that were negative for bacterial microorganisms. Stains of the colonies demonstrated clusters of viable neoplastic cells.

Neonatal meningoencephalitis caused by Bacillus cereus.

The classic organisms associated with central nervous system infection in the neonate are herpes simplex, Listeria monocytogenes, Escherichia coli, and Streptococcus agalactiae; we describe an unusual case of neonatal meningoencephalitis caused by Bacillus cereus.