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The human and organizational burdens of sepsis can be reduced by early recognition, diagnosis, therapy, and systems that support more effective management. The World Health Organization's (WHO's) 2020 Global Report on the Epidemiology and Burden of Sepsis will add importance to the clinical laboratory's role of monitoring blood culture metrics. The report details strategies for future efforts to characterize the global impact of sepsis more accurately, to improve the characterization of and differences in epidemiology, and to make estimates of disease burden more accurate. As the WHO is working toward improved global sepsis monitoring, re-assessment of the way clinical laboratories can contribute to this effort are long overdue at the local, national, and global levels. Clinical laboratory dogma describes the importance of blood cultures (BCs) and optimization of BC processes, with documented impact on patient care. The BC is considered a first-line investigation for the microbiological diagnosis of sepsis, bacteremia, and fungemia and can detect a wide variety of microorganisms at a relatively low cost. Once microbes are detected in BC broth, subcultures yield microbial colonies for subsequent identification and antimicrobial susceptibility testing, and information is incorporated into the final BC result. However, the impact of BCs and the speed at which BCs are collected, incubated, and reported are minimized in the “surviving sepsis” literature, except for BC collection prior to the administration of antibiotics. Most hospital quality efforts focus on compliance with the 1-, 3-, and 6-h sepsis bundles with limited focus on the contribution of microbiology's role in rapid incubation, pathogen reporting, and other diagnostics that drive antimicrobial de-escalation and escalation protocols. Clinical microbiology and laboratory medicine bear responsibility to ensure that BCs are collected properly and in a timely manner; that they are rapidly transported to the laboratory and incubated quickly; and that results are achieved and reported in a timely, reproducible, and accurate fashion. The quality metrics we discuss are not all required by inspection agencies; however, considering the initiatives to reduce the human cost of sepsis, attention to best practices is useful to optimize BC performance and BC yield. Examples of blood culture quality monitors and metrics are listed for laboratory consideration. Each laboratory may have unique situations in which one or more of the monitors may prove to be useful.

In January 2020, a cluster of pneumonia cases was reported in Wuhan, China. A global pandemic followed. The infection, called novel coronavirus disease 2019 (COVID-19), is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Common symptoms of COVID-19 illness included fever, cough, and abnormal findings on chest computed tomography. Nucleic acid testing, in the form of real-time reverse transcriptase polymerase chain reaction, is essential for diagnosing COVID-19 from respiratory samples from infected patients. Still, many questions remain surrounding the optimization of pre-analytical factors, such as specimen selection, collection, and transport. This review summarizes the current publications that describe viral density and specimen suitability for molecular detection methods. Of note, many of the reports represent studies with small sample sizes, and information may change as more is learned about specimen types as the pandemic continues.

Because rapid and accurate blood culture (BC)2 results are critical for positive patient outcomes, laboratories continue to adopt quality indicators and metrics, best practices, and new methodologies to improve BC performance (1). As new technologies are implemented, laboratories may struggle with increased workflow complexity. Moreover, as the healthcare landscape changes, laboratories are faced with how to operationally manage BCs throughout their healthcare organization (HCO). In this article, the management of BCs in a large integrated laboratory system will be discussed, with a focus on our telemicroscopy program that provides added accuracy and …

The incidence of tick-borne human infectious syndromes has dramatically risen in the last 2 decades, leading to significant morbidity and mortality. The importance of a rapid and accurate diagnosis to distinguish anaplasmosis from other febrile illnesses is paramount for the prompt administration of appropriate antibiotics and is imperative for the successful treatment of the tick-borne infections. We summarize the current literature describing the biology, clinical presentation, and epidemiology of human granulocytic anaplasmosis. In addition, we report a case of human granulocytic anaplasmosis in one of the less common geographical areas (Pennsylvania) during the winter month of December. Laboratory methods, including use of an acridine orange buffy coat fluorescent stain that helped decreased the time to result and increased confidence in the diagnosis, are also described.

Formalin-fixed, paraffin-embedded tissue is regarded as a valuable source of clinical information. Due to its potential use in biomarker discovery, research initiatives are driving the implementation of best practices in pre-analytical and post-analytical processing. The clinical microbiology laboratory stands to gain from these efforts to integrate standard operating procedures, extraction methods, and quality metrics. As molecular diagnostics broadens its test menu for tissue samples, results derived from tissue will become more reproducible, and microbiological detection methods, in the form of in-situ hybridization, polymerase chain reaction, and nucleic acid sequencing, are likely to become more commonplace. Although limited in number, infectious disease diagnostic options are available for fixed tissue. Thus, it is important for microbiologists to better understand histological practices and the underpinnings of the tissue processing that occurs prior to the time of nucleic acid amplification attempts.

Herpes simplex virus (HSV) oral shedding has not been studied among HIV-positive children in Africa. We sought to evaluate longitudinal oral HSV reactivation in HIV-positive and -negative children. Twenty HIV-positive antiretroviral-naive and 10 HIV-negative children aged 3–12 years in Tanzania were followed prospectively for 14 days. Oral swabs were collected daily and submitted for HSV DNA PCR analysis. Clinical data were collected via chart review and daily diaries. HSV DNA was detected in 10 (50%) of HIV-positive and 4 (40%) of HIV-negative children. Children who shed HSV had virus detected in a median of 21.4% of samples; shedding was intermittent. Median CD4 count among HIV-infected children was 667 cells/µL in those with positive HSV DNA and 886 cells/µL in those who were negative ( p = 0.6). Of the HIV-positive children reporting prior sores, five (83%) had positive HSV swabs, whereas the one HIV-negative child with prior sores did not have a PCR-positive swab. HSV is detected frequently in children with and without HIV. HIV-infected children reporting oral sores have a high rate of HSV detection. Given the proven strong interactions between HIV and HSV, further study of co-infection with these viruses is warranted in children.

The identification of organisms from positive blood cultures generally takes several days. However, recently developed rapid diagnostic methods offer the potential for organism identification within only a few hours of blood culture positivity. In this study, we evaluated the performance of three commercial methods to rapidly identify organisms directly from positive blood cultures: Quick FISH (AdvanDx, Wolburn, MA), Verigene Gram-Positive Blood Culture (BC-GP; Nanosphere, Northbrook, IL), and matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) with Sepsityper processing (Bruker Daltonics, Billerica, MA). A total of 159 blood cultures (VersaTREK Trek Diagnostic Systems, Cleveland, OH) positive for Gram-positive and Gram-negative bacteria as well as yeast were analyzed with Quick FISH and MALDI-TOF MS. In all, 102 blood cultures were analyzed using the BC-GP assay. For monomicrobial cultures, we observed 98.0% concordance with routine methods for both Quick FISH (143/146) and the BC-GP assay (93/95). MALDI-TOF MS demonstrated 80.1% (117/146) and 87.7% (128/146) concordance with routine methods to the genus and species levels, respectively. None of the methods tested were capable of consistently identifying polymicrobial cultures in their entirety or reliably differentiating Streptococcus pneumoniae from viridans streptococci. Nevertheless, the methods evaluated in this study are convenient and accurate for the most commonly encountered pathogens and have the potential to dramatically reduce turnaround time for the provision of results to the treating physician.

Biorepositories aim to acquire, authenticate, preserve, develop, and distribute biological specimens, along with information, technology, intellectual property, and standards for the advancement and application of scientific knowledge. Biorepositories are critical components of modern healthcare , as they provide resources to advance translational biomedical research, navigation through the FDA process for in vitro devices, verification and validation of diagnostic methods, and personalized medicine. In order to fully realize the opportunities that biorepositories offer to medicine, laboratories must be able to guarantee high-quality biospecimens, associated information, and a system able to respond to the needs of method verification, quality control, or the requests of scientific colleagues. A biorepository requires funding resources and space, which are often difficult to find within existing health care resources, yet health care may benefit most from the resources biorepositories provide. It is crucial that institutions be able to recognize these issues and start preparing and releasing adequate resources to support the development of biorepositories with a common goal to improve health care by increasing the quality of biomedical research and diagnostic test development. The future of biorepositories depends on the development of more evidence-based practices in both the research and clinical settings. As the field matures, educating a new generation of biospecimen/biobanking scientists will be an important need.

Lactobacillus spp. are part of the normal human flora and are generally assumed to be nonpathogenic. We determined the genotypic identification of >100 Lactobacillus isolates from clinical specimens in the context of presumed pathogenic potential (e.g., recovered as the single/predominant isolate from a sterile site or at ≥10 5 CFU/ml from urine). This study assessed the clinical significance and the frequency of occurrence of each Lactobacillus sp. We identified 16 species of Lactobacillus by 16S rRNA gene sequence analysis, 10 of which could not be associated with disease. While Lactobacillus rhamnosus , Lactobacillus gasseri , and Lactobacillus paracasei were associated with infections, L. gasseri was also a common colonizing/contaminating species. Lactobacillus casei , Lactobacillus johnsonii , and Lactobacillus delbrueckii were associated with at least one infection. Species commonly used in probiotic products (e.g., L. rhamnosus and L. casei ) were identical, by 16S rRNA gene sequencing, to our isolates associated with disease. Human isolates of Lactobacillus spp. have differing site associations and levels of clinical significance. Knowing the niche and pathogenic potential of each Lactobacillus sp. can be of importance to both clinical microbiology and the food and probiotic supplement industry.

The immunocompromised host is a particularly vulnerable population in whom routine and unusual infections can easily and frequently occur. Prosthetic devices are commonly used in these patients and the infections associated with those devices present a number of challenges for both the microbiologist and the clinician. Biofilms play a major role in device-related infections, which may contribute to failed attempts to recover organisms from routine culture methods. Moreover, device-related microorganisms can be difficult to eradicate by antibiotic therapy alone. Changes in clinical practice and advances in laboratory diagnostics have provided significant improvements in the detection and accurate diagnosis of device-related infections. Disruption of the bacterial biofilm plays an essential role in recovering the causative agent in culture. Various culture and nucleic acid amplification techniques are more accurate to guide directed treatment regimens. This chapter reviews the performance characteristics of currently available diagnostic assays and summarizes published guidelines, where available, for addressing suspected infected prosthetic devices.

Bacteremia and sepsis are conditions associated with high mortality and are of great impact to health care operations. Among the top causes of mortality in the United States, these conditions cause over 600 fatalities each day. Empiric, broad-spectrum treatment is a common but often a costly approach that may fail to effectively target the correct microbe, may inadvertently harm patients via antimicrobial toxicity or downstream antimicrobial resistance. To meet the diagnostic challenges of bacteremia and sepsis, laboratories must understand the complexity of diagnosing and treating septic patients, in order to focus on creating algorithms that can help direct a more targeted approach to antimicrobial therapy and synergize with existing clinical practices defined in new Surviving Sepsis Guidelines. Significant advances have been made in improving blood culture media; as yet no molecular or antigen-based method has proven superior for the detection of bacteremia in terms of limit of detection. Several methods for rapid molecular identification of pathogens from blood cultures bottles are available and many more are on the diagnostic horizon. Ultimately, early intervention by molecular detection of bacteria and fungi directly from whole blood could provide the most patient benefit and contribute to tailored antibiotic coverage of the patient early on in the course of the disease. Although blood cultures remain as the best means of diagnosing bacteremia and candidemia, complementary testing with antigen tests, microbiologic investigations from other body sites, and histopathology can often aid in the diagnosis of disseminated disease, and application of emerging nucleic acid test methods and other new technology may greatly impact our ability to bacteremic and septic patients, particularly those who are immunocompromised.

Group B Streptococci (GBS) are ß-hemolytic, gram-positive bacteria that are typically associated with infections in human newborns or immunocompromised adults. However, mutation in the two-component regulator CovR/S relieves repression of hemolysin, potentially increasing virulence of GBS. We report the isolation of hyperhemolytic/hyperpigmented GBS strain from an adolescent patient who presented to the University of Washington clinic with symptoms of sore throat. While the patient also tested positive for mononucleosis, a GBS strain with increased hemolysis was isolated from the throat swab obtained from the patient. As hyperhemolytic/hyperpigmented GBS strains are typically associated with mutations in the regulator CovR/CovS, we sequenced the

Vibrio cholerae is highly motile by the action of a single polar flagellum. The loss of motility reduces the infectivity of V. cholerae , demonstrating that motility is an important virulence factor. FlrC is the sigma-54-dependent positive regulator of flagellar genes. Recently, the genes VC2206 ( flgP ) and VC2207 ( flgO ) were identified as being regulated by FlrC via a microarray analysis of an flrC mutant (D. C. Morris, F. Peng, J. R. Barker, and K. E. Klose, J. Bacteriol. 190:231-239, 2008). FlgP is reported to be an outer membrane lipoprotein required for motility that functions as a colonization factor. The study reported here focuses on the characterization of flgO , the first gene in the flgOP operon. We show that FlgO and FlgP are important for motility, as strains with mutations in the flgOP genes have reduced motility phenotypes. The flgO and flgP mutant populations display fewer motile cells as well as reduced numbers of flagellated cells. The flagella produced by the flgO and flgP mutant strains are shorter in length than the wild-type flagella, which can be restored by inhibiting rotation of the flagellum. FlgO is an outer membrane protein that localizes throughout the membrane and not at the flagellar pole. Although FlgO and FlgP do not specifically localize to the flagellum, they are required for flagellar stability. Due to the nature of these motility defects, we established that the flagellum is not sufficient for adherence; rather, motility is the essential factor required for attachment and thus colonization by V. cholerae O1 of the classical biotype. This study reveals a novel mechanism for which the outer membrane proteins FlgO and FlgP function in motility to mediate flagellar stability and influence attachment and colonization.

Cochliomyia hominivorax, the New World screwworm, was a serious livestock pest in the southern United States until the 1960s, when it was successfully eradicated by the release of sterile male flies. It remains endemic in parts of the Caribbean and South America, and there is concern that climate change may extend its geographic distribution. Cochliomyia hominivorax is voracious and can cause extensive damage to soft tissue and bone. We describe the case of a 26-year-old traveler who presented with otalgia and bloody otorrhea after returning from a vacation in the Dominican Republic, where exposure to screwworm flies most likely occurred during a nap on the beach. The causative agent was recognized by its characteristic larval anatomy, which includes pigmented dorsal tracheal trunks and posterior spiracles with an open peritreme.

Vibrio cholerae is a Gram-negative enteric pathogen. This unit includes protocols for the growth and maintenance of V. cholerae in the laboratory. Curr. Protoc. Microbiol. 17:6A.1.1-6A.1.7. © 2010 by John Wiley & Sons, Inc. Keywords: Vibrio cholerae; laboratory growth; cholera

Flagellar motility has long been regarded as an important virulence factor. In Vibrio cholerae , the single polar flagellum is essential for motility as well as for proper attachment and colonization. In this study, we demonstrate that the novel flagellar protein FlgT is involved in anchoring the flagellum to the V. cholerae cell. A screen for novel colonization factors by use of Tn phoA mutagenesis identified flgT . An in-frame deletion of flgT established that FlgT is required for attachment, colonization, and motility. Transmission electron microscopy revealed that while the flgT mutant is capable of assembling a phenotypically normal flagellum, the flgT population is mostly aflagellate compared to the wild-type population. Further analyses indicated that the flagellum of the flgT mutant is released into the culture supernatant from the cell upon completion of assembly. Additionally, hook basal body complexes appear to be released along with the filament. These results indicate that FlgT functions to stabilize the flagellar apparatus at the pole of the cell.

Vibrio cholerae is the etiologic agent of cholera in humans. Intestinal colonization occurs in a stepwise fashion, initiating with attachment to the small intestinal epithelium. This attachment is followed by expression of the toxin-coregulated pilus, microcolony formation, and cholera toxin (CT) production. We have recently characterized a secreted attachment factor, GlcNAc binding protein A (GbpA), which functions in attachment to environmental chitin sources as well as to intestinal substrates. Studies have been initiated to define the regulatory network involved in GbpA induction. At low cell density, GbpA was detected in the culture supernatant of all wild-type (WT) strains examined. In contrast, at high cell density, GbpA was undetectable in strains that produce HapR, the central regulator of the cell density-dependent quorum-sensing system of V. cholerae . HapR represses the expression of genes encoding regulators involved in V. cholerae virulence and activates the expression of genes encoding the secreted proteases HapA and PrtV. We show here that GbpA is degraded by HapA and PrtV in a time-dependent fashion. Consistent with this, Δ hapA Δ prtV strains attach to chitin beads more efficiently than either the WT or a Δ hapA Δ prtV Δ gbpA strain. These results suggest a model in which GbpA levels fluctuate in concert with the bacterial production of proteases in response to quorum-sensing signals. This could provide a mechanism for GbpA-mediated attachment to, and detachment from, surfaces in response to environmental cues.

We reported previously that low concentrations of sodium citrate strongly promote biofilm formation by Staphylococcus aureus laboratory strains and clinical isolates. Here, we show that citrate promotes biofilm formation via stimulating both cell-to-surface and cell-to-cell interactions. Citrate-stimulated biofilm formation is independent of the ica locus, and in fact, citrate represses polysaccharide adhesin production. We show that fibronectin binding proteins FnbA and FnbB and the global regulator SarA, which positively regulates fnbA and fnbB gene expression, are required for citrate's positive effects on biofilm formation, and citrate also stimulates fnbA and fnbB gene expression. Biofilm formation is also stimulated by several other tricarboxylic acid (TCA) cycle intermediates in an FnbA-dependent fashion. While aconitase contributes to biofilm formation in the absence of TCA cycle intermediates, it is not required for biofilm stimulation by these compounds. Furthermore, the GraRS two-component regulator and the GraRS-regulated efflux pump VraFG, identified for their roles in intermediate vancomycin resistance, are required for citrate-stimulated cell-to-cell interactions, but the GraRS regulatory system does not impact the expression of the fnbA and fnbB genes. Our data suggest that distinct genetic factors are required for the early steps in citrate-stimulated biofilm formation. Given the role of FnbA/FnbB and SarA in virulence in vivo and the lack of a role for ica -mediated biofilm formation in S. aureus catheter models of infection, we propose that the citrate-stimulated biofilm formation pathway may represent a clinically relevant pathway for the formation of these bacterial communities on medical implants.

Background. Microbial biofilms form on central venous catheters and may be associated with systemic infections as well as decreased dialysis efficiency due to catheter thrombosis. The most widely used anticoagulant catheter lock solution in the US is sodium heparin. We have previously shown that sodium heparin in clinically relevant concentrations enhances Staphylococcus aureus biofilm formation. In the present study, we examine the effect of several alternative catheter lock solutions on in vitro biofilm formation by laboratory and clinical isolates of S. aureus and coagulase-negative staphylococci (CNS). Methods. Lepirudin, low molecular weight heparin, tissue plasminogen activator, sodium citrate, sodium citrate with gentamicin and sodium ethylene diamine tetra-acetic acid (EDTA) were assessed for their effect on biofilm formation on polystyrene, polyurethane and silicon elastomer. Results. Sodium citrate at concentrations above 0.5% efficiently inhibits biofilm formation and cell growth of S. aureus and Staphylococcus epidermidis. Subinhibitory concentrations of sodium citrate significantly stimulate biofilm formation in most tested S. aureus strains, but not in CNS strains. Sodium EDTA was effective in prevention of biofilm formation as was a combination of sodium citrate and gentamicin. Low molecular weight heparin stimulated biofilm formation of S. aureus, while lepirudin and tissue plasminogen activator had little effect on S. aureus biofilm formation. Conclusions. This in vitro study demonstrates that heparin alternatives, sodium citrate and sodium EDTA, can prevent the formation of S. aureus biofilms, suggesting that they may reduce the risk of biofilm-associated complications in indwelling catheters. This finding suggests a biological mechanism for the observed improvement in catheter-related outcomes in recent clinical comparisons of heparin and trisodium citrate as catheter locking solutions. A novel and potential clinically relevant finding of the present study is the observation that citrate at low levels strongly stimulates biofilm formation by S. aureus.