Cathy A. Petti

Laboratory Medicine in Africa: A Barrier to Effective Health Care

Providing health care in sub-Saharan Africa is a complex problem. Recent reports call for more resources to assist in the prevention and treatment of infectious diseases that affect this population, but policy makers, clinicians, and the public frequently fail to understand that diagnosis is essential to the prevention and treatment of disease. Access to reliable diagnostic testing is severely limited in this region, and misdiagnosis commonly occurs. Understandably, allocation of resources to diagnostic laboratory testing has not been a priority for resource-limited health care systems, but unreliable and inaccurate laboratory diagnostic testing leads to unnecessary expenditures in a region already plagued by resource shortages, promotes the perception that laboratory testing is unhelpful, and compromises patient care. We explore the barriers to implementing consistent testing within this region and illustrate the need for a more comprehensive approach to the diagnosis of infectious diseases, with an emphasis on making laboratory testing a higher priority.

Impaired neutrophil extracellular trap (NET) formation: a novel innate immune deficiency of human neonates

Neutrophils are highly specialized innate effector cells that have evolved for killing of pathogens. Human neonates have a common multifactorial syndrome of neutrophil dysfunction that is incompletely characterized and contributes to sepsis and other severe infectious complications. We identified a novel defect in the antibacterial defenses of neonates: inability to form neutrophil extracellular traps (NETs). NETs are lattices of extracellular DNA, chromatin, and antibacterial proteins that mediate extracellular killing of microorganisms and are thought to form via a unique death pathway signaled by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-generated reactive oxygen species (ROS). We found that neutrophils from term and preterm infants fail to form NETs when activated by inflammatory agonists-in contrast to leukocytes from healthy adults. The deficiency in NET formation is paralleled by a previously unrecognized deficit in extracellular bacterial killing. Generation of ROSs did not complement the defect in NET formation by neonatal neutrophils, as it did in adult cells with inactivated NADPH oxidase, demonstrating that ROSs are necessary but not sufficient signaling intermediaries and identifying a deficiency in linked or downstream pathways in neonatal leukocytes. Impaired NET formation may be a critical facet of a common developmental immunodeficiency that predisposes newborn infants to infection.

Detection and Identification of Microorganisms by Gene Amplification and Sequencing

Gene amplification and sequencing have led to the discovery of new pathogens as agents of disease and have enabled us to better classify microorganisms from culture. Sequence-based identification of bacteria and fungi using culture is more objective and accurate than conventional methods, especially for classifying unusual microorganisms that are emerging pathogens in immunocompromised hosts. Although a powerful tool, the interpretation of sequence-based classification can be challenging as microbial taxonomy grows more complex, without known clinical correlatives. Additionally, broad-range gene polymerase chain reaction and sequencing have emerged as alternative, culture-independent methods for detecting pathogens from clinical material. The promise of this technique has remained strong, limited mainly by contamination and inadequate sensitivity issues. This review explains sequence-based microbial classification, with emphasis on relating the complex world of microbial taxonomy to a clinical context. Additionally, this review discusses a rational approach to broad-range bacterial polymerase chain reaction and gene sequencing when applied directly to clinical samples.

The Role of 16S rRNA Gene Sequencing in Identification of Microorganisms Misidentified by Conventional Methods

ABSTRACT Traditional methods for microbial identification require the recognition of differences in morphology, growth, enzymatic activity, and metabolism to define genera and species. Full and partial 16S rRNA gene sequencing methods have emerged as useful tools for identifying phenotypically aberrant microorganisms. We report on three bacterial blood isolates from three different College of American Pathologists-certified laboratories that were referred to ARUP Laboratories for definitive identification. Because phenotypic identification suggested unusual organisms not typically associated with the submitted clinical diagnosis, consultation with the Medical Director was sought and further testing was performed including partial 16S rRNA gene sequencing. All three patients had endocarditis, and conventional methods identified isolates from patients A, B, and C as a Facklamia sp., Eubacterium tenue, and a Bifidobacterium sp. 16S rRNA gene sequencing identified the isolates as Enterococcus faecalis, Cardiobacterium valvarum, and Streptococcus mutans, respectively. We conclude that the initial identifications of these three isolates were erroneous, may have misled clinicians, and potentially impacted patient care. 16S rRNA gene sequencing is a more objective identification tool, unaffected by phenotypic variation or technologist bias, and has the potential to reduce laboratory errors.

Role of the Laboratory in Diagnosis of Influenza during Seasonal Epidemics and Potential Pandemics

Laboratory diagnosis of influenza is critical to its treatment and surveillance. With the emergence of novel and highly pathogenic avian influenza viruses, the role of the laboratory has been further extended to include isolation and subtyping of the virus to monitor its appearance and facilitate appropriate vaccine development. Recent progress in enhancing testing for influenza promises to both improve the management of patients with influenza and decrease associated health care costs. The present review covers the technological characteristics and utilization features of currently available diagnostic tests, the factors that influence the selection of such tests, and the developments that are essential for pandemic preparedness.

Staphylococcus aureus bacteremia and endocarditis.

Staphylococcus aureus is a leading cause of bacteremia and endocarditis. Over the past several years, the frequency of S. aureus bacteremia (SAB) has increased dramatically. This increasing frequency, coupled with increasing rates of antibiotic resistance, has renewed interest in this serious, common infection. S. aureus is a unique pathogen because of its virulent properties, its protean manifestations, and its ability to cause endocarditis on architecturally normal cardiac valves. Although the possibility of underlying endocarditis arises in virtually every patient with SAB, only a minority of bacteremic patients will actually have cardiac involvement. Distinguishing patients with S. aureus infective endocarditis (IE) from those with uncomplicated SAB is essential, but often difficult. In this review, the authors summarize recent changes in the epidemiology of SAB and IE, discuss the challenges in distinguishing SAB from IE, and discuss current trends in the management of patients with SAB and IE.

Identification of an Emerging Pathogen, Mycobacterium massiliense, by rpoB Sequencing of Clinical Isolates Collected in the United States

ABSTRACT Mycobacterium massiliense is a rapidly growing mycobacterium that is indistinguishable from Mycobacterium chelonae/M. abscessus by partial 16S rRNA gene sequencing. We sequenced rpoB, sodA, and hsp65 genes from isolates previously identified as being M. chelonae/M. abscessus and identified M. massiliense from isolates from two patients with invasive disease representing the first reported cases in the United States.

Utility of Extended Blood Culture Incubation for Isolation of Haemophilus, Actinobacillus, Cardiobacterium, Eikenella, and Kingella Organisms: a Retrospective Multicenter Evaluation

ABSTRACT The incidence of and average time to detection for Haemophilus, Actinobacillus, Cardiobacterium, Eikenella, and Kingella (HACEK) bacteria in blood cultures with standard incubation and the utility of extended incubation of blood culture bottles were reviewed at four tertiary care microbiology laboratories. HACEK organisms were isolated from 35 (<0.005%) of 59,203 positive blood cultures. None of 407 blood cultures with extended incubation grew HACEK or other bacteria. Bacteremia from HACEK bacteria is rare, and extended incubation of blood cultures to recover HACEK bacteria is unnecessary.

Application of SmartGene IDNS Software to Partial 16S rRNA Gene Sequences for a Diverse Group of Bacteria in a Clinical Laboratory

ABSTRACT Laboratories often receive clinical isolates for bacterial identification that have ambiguous biochemical profiles by conventional testing. With the emergence of 16S rRNA gene sequencing as an identification tool, we evaluated the usefulness of SmartGene IDNS, a 16S rRNA sequence database and software program for microbial identification. Identification by conventional methods of a diverse group of bacterial clinical isolates was compared with gene sequences interrogated by the SmartGene and MicroSeq databases. Of 300 isolates, SmartGene identified 295 (98%) to the genus level and 262 (87%) to the species level, with 5 (2%) being inconclusive. MicroSeq identified 271 (90%) to the genus level and 223 (74%) to the species level, with 29 (10%) being inconclusive. SmartGene and MicroSeq agreed on the genus for 233 (78%) isolates and the species for 212 (71%) isolates. Conventional methods identified 291 (97%) isolates to the genus level and 208 (69%) to the species level, with 9 (3%) being inconclusive. SmartGene, MicroSeq, and conventional identifications agreed for 193 (64%) of the results. Twenty-seven microorganisms were not represented in MicroSeq, compared to only 2 not represented in SmartGene. Overall, SmartGene IDNS provides comprehensive and accurate identification of a diverse group of bacteria and has the added benefit of being a user-friendly program that can be modified to meet the unique needs of clinical laboratories.

Gordonia Species: Emerging Pathogens in Pediatric Patients That Are Identified by 16S Ribosomal RNA Gene Sequencing

Gordonia species are emerging pathogens that are often misidentified as Rhodococcus or Nocardia species but are reliably distinguished by 16S ribosomal RNA gene sequencing. We present a case series of 6 episodes of catheter-associated infection caused by Gordonia species in 5 patients seen at a tertiary care pediatric hospital and describe the management and outcomes of this infection in adults and children.