Christopher R. Polage

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.

Overdiagnosis of Clostridium difficile Infection in the Molecular Test Era

IMPORTANCE Clostridium difficile is a major cause of health care-associated infection, but disagreement between diagnostic tests is an ongoing barrier to clinical decision making and public health reporting. Molecular tests are increasingly used to diagnose C difficile infection (CDI), but many molecular test-positive patients lack toxins that historically defined disease, making it unclear if they need treatment. OBJECTIVE To determine the natural history and need for treatment of patients who are toxin immunoassay negative and polymerase chain reaction (PCR) positive (Tox-/PCR+) for CDI. DESIGN, SETTING, AND PARTICIPANTS Prospective observational cohort study at a single academic medical center among 1416 hospitalized adults tested for C difficile toxins 72 hours or longer after admission between December 1, 2010, and October 20, 2012. The analysis was conducted in stages with revisions from April 27, 2013, to January 13, 2015. MAIN OUTCOMES AND MEASURES Patients undergoing C difficile testing were grouped by US Food and Drug Administration-approved toxin and PCR tests as Tox+/PCR+, Tox-/PCR+, or Tox-/PCR-. Toxin results were reported clinically. Polymerase chain reaction results were not reported. The main study outcomes were duration of diarrhea during up to 14 days of treatment, rate of CDI-related complications (ie, colectomy, megacolon, or intensive care unit care) and CDI-related death within 30 days. RESULTS Twenty-one percent (293 of 1416) of hospitalized adults tested for C difficile were positive by PCR, but 44.7% (131 of 293) had toxins detected by the clinical toxin test. At baseline, Tox-/PCR+ patients had lower C difficile bacterial load and less antibiotic exposure, fecal inflammation, and diarrhea than Tox+/PCR+ patients (P < .001 for all). The median duration of diarrhea was shorter in Tox-/PCR+ patients (2 days; interquartile range, 1-4 days) than in Tox+/PCR+ patients (3 days; interquartile range, 1-6 days) (P = .003) and was similar to that in Tox-/PCR- patients (2 days; interquartile range, 1-3 days), despite minimal empirical treatment of Tox-/PCR+ patients. No CDI-related complications occurred in Tox-/PCR+ patients vs 10 complications in Tox+/PCR+ patients (0% vs 7.6%, P < .001). One Tox-/PCR+ patient had recurrent CDI as a contributing factor to death within 30 days vs 11 CDI-related deaths in Tox+/PCR+ patients (0.6% vs 8.4%, P = .001). CONCLUSIONS AND RELEVANCE Among hospitalized adults with suspected CDI, virtually all CDI-related complications and deaths occurred in patients with positive toxin immunoassay test results. Patients with a positive molecular test result and a negative toxin immunoassay test result had outcomes that were comparable to patients without C difficile by either method. Exclusive reliance on molecular tests for CDI diagnosis without tests for toxins or host response is likely to result in overdiagnosis, overtreatment, and increased health care costs.

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.

Nosocomial Diarrhea: Evaluation and Treatment of Causes Other Than Clostridium difficile

Diarrhea is common among hospitalized patients but the causes are distinct from those of diarrhea in the community. We review existing data about the epidemiology of nosocomial diarrhea and summarize recent progress in understanding the mechanisms of diarrhea. Clinicians should recognize that most cases of nosocomial diarrhea have a noninfectious etiology, including medications, underlying illness, and enteral feeding. Apart from Clostridium difficile, the frequency of infectious causes such as norovirus and toxigenic strains of Clostridium perfringens, Klebsiella oxytoca, Staphylococcus aureus, and Bacteroides fragilis remains largely undefined and test availability is limited. Here we provide a practical approach to the evaluation and management of nosocomial diarrhea when tests for C. difficile are negative.

Identification of Gram-Negative Bacteria and Genetic Resistance Determinants from Positive Blood Culture Broths by Use of the Verigene Gram-Negative Blood Culture Multiplex Microarray-Based Molecular Assay

ABSTRACT Bloodstream infection is a serious condition associated with significant morbidity and mortality. The outcome of these infections can be positively affected by the early implementation of effective antibiotic therapy based on the identification of the infecting organism and genetic markers associated with antibiotic resistance. In this study, we evaluated the microarray-based Verigene Gram-negative blood culture (BC-GN) assay in the identification of 8 genus or species targets and 6 genetic resistance determinants in positive blood culture broths. A total of 1,847 blood cultures containing Gram-negative organisms were tested using the BC-GN assay. This comprised 729 prospective fresh, 781 prospective or retrospective frozen, and 337 simulated cultures representing 7 types of aerobic culture media. The results were compared to those with standard bacterial culture and biochemical identification with nucleic acid sequence confirmation of the resistance determinants. Among monomicrobial cultures, the positive percent agreement (PPA) of the BC-GN assay with the reference method was as follows; Escherichia coli, 100%; Klebsiella pneumoniae, 92.9%; Klebsiella oxytoca, 95.5%; Enterobacter spp., 99.3%; Pseudomonas aeruginosa, 98.9%; Proteus spp., 100%; Acinetobacter spp., 98.4%; and Citrobacter spp., 100%. All organism identification targets demonstrated >99.5% negative percent agreement (NPA) with the reference method. Of note, 25/26 cultures containing K. pneumoniae that were reported as not detected by the BC-GN assay were subsequently identified as Klebsiella variicola. The PPA for identification of resistance determinants was as follows; bla CTX-M, 98.9%; bla KPC, 100%; bla NDM, 96.2%; bla OXA, 94.3%; bla VIM, 100%; and bla IMP, 100%. All resistance determinant targets demonstrated >99.9% NPA. Among polymicrobial specimens, the BC-GN assay correctly identified at least one organism in 95.4% of the broths and correctly identified all organisms present in 54.5% of the broths. The sample-to-result processing and automated reading of the detection microarray results enables results within 2 h of culture positivity.

Effect of Cefazolin Treatment on the Nonresonant Raman Signatures of the Metabolic State of Individual Escherichia coli Cells

Laser tweezers Raman spectroscopy (LTRS) was used to characterize the Raman fingerprints of the metabolic states of Escherichia coli (E. coli) cells and to determine the spectral changes associated with cellular response to the antibiotic Cefazolin. The Raman spectra of E. coli cells sampled at different time points in the bacterial growth curve exhibited several spectral features that enabled direct identification of the growth phase of the bacteria. Four groups of Raman peaks were identified based on similarities in the time-dependent behavior of their intensities over the course of the growth curve. These groupings were also consistent with the different biochemical species represented by the Raman peaks. Raman peaks associated with DNA and RNA displayed a decrease in intensity over time, while protein-specific Raman vibrations increased at different rates. The adenine ring-breathing mode at 729 and the 1245 cm(-1) vibration peaked in intensity within the first 10 h and decreased afterward. Application of principal component analysis (PCA) to the Raman spectra enabled accurate identification of the different metabolic states of the bacterial cells. The Raman spectra of cells exposed to Cefazolin at the end of log phase exhibited a different behavior. The 729 and 1245 cm(-1) Raman peaks showed a slight decrease in intensity from 4 to 10 h after inoculation. Moreover, a shift in the spectral position of the adenine ring-breathing mode from 724 to 729 cm(-1), which was observed during normal bacterial growth, was inhibited during antibiotic drug treatment. These results suggest that potential Raman markers exist that can be used to identify E. coli cell response to antibiotic drug treatment.

Assessment of the Utility of Viral Culture of Cerebrospinal Fluid

Nucleic acid amplification testing is the preferred method to detect enteroviruses and Herpesviridae in cerebrospinal fluid, but clinicians still request viral culture. Review of 22,394 viral cultures of cerebrospinal fluid samples found that <0.1% recovered nonenterovirus, non-Herpesviridae species, suggesting that, when nucleic acid amplification testing is performed, viral culture may have no additional benefit.

Outcomes in patients tested for Clostridium difficile toxins

Clostridium difficile testing is shifting from toxin detection to C. difficile detection. Yet, up to 60% of patients with C. difficile by culture test negative for toxins and it is unclear whether they are infected or carriers. We reviewed medical records for 7046 inpatients with a C. difficile toxin test from 2005 to 2009 to determine the duration of diarrhea and rate of complications and mortality among toxin-positive (toxin+) and toxin- patients. Overall, toxin- patients had less severe diarrhea, fewer diarrhea days, and lower mortality (P < 0.001, all comparisons) than toxin+ patients. One toxin- patient (n = 1/6121; 0.02%) was diagnosed with pseudomembranous colitis, but there were no complications such as megacolon or colectomy for fulminant CDI among toxin- patients. These data suggest that C. difficile-attributable complications are rare among patients testing negative for C. difficile toxins. More studies are needed to evaluate the clinical significance of C. difficile detection in toxin- patients.

Respiratory syncytial virus outbreak in a long-term care facility detected using reverse transcriptase polymerase chain reaction: an argument for real-time detection methods.

OBJECTIVES: To report an outbreak of respiratory synctyial virus (RSV) in a long‐term care facility (LTCF) during ongoing routine respiratory illness surveillance.

Evaluation of Escherichia coli Cell Response to Antibiotic Treatment by Use of Raman Spectroscopy with Laser Tweezers

ABSTRACT Laser tweezers Raman spectroscopy was used to detect the cellular response of Escherichia coli cells to penicillin G-streptomycin and cefazolin. Time-dependent intensity changes of several Raman peaks at 729, 1,245, and 1,660 cm−1 enabled untreated cells and cells treated with the different antibiotic drugs to be distinguished.