Carey-Ann D. Burnham

Diagnosis of Clostridium difficile Infection: an Ongoing Conundrum for Clinicians and for Clinical Laboratories

SUMMARY Clostridium difficile is a formidable nosocomial and community-acquired pathogen, causing clinical presentations ranging from asymptomatic colonization to self-limiting diarrhea to toxic megacolon and fulminant colitis. Since the early 2000s, the incidence of C. difficile disease has increased dramatically, and this is thought to be due to the emergence of new strain types. For many years, the mainstay of C. difficile disease diagnosis was enzyme immunoassays for detection of the C. difficile toxin(s), although it is now generally accepted that these assays lack sensitivity. A number of molecular assays are commercially available for the detection of C. difficile. This review covers the history and biology of C. difficile and provides an in-depth discussion of the laboratory methods used for the diagnosis of C. difficile infection (CDI). In addition, strain typing methods for C. difficile and the evolving epidemiology of colonization and infection with this organism are discussed. Finally, considerations for diagnosing C. difficile disease in special patient populations, such as children, oncology patients, transplant patients, and patients with inflammatory bowel disease, are described. As detection of C. difficile in clinical specimens does not always equate with disease, the diagnosis of C. difficile infection continues to be a challenge for both laboratories and clinicians.

Mupirocin and Chlorhexidine Resistance in Staphylococcus aureus in Patients with Community-Onset Skin and Soft Tissue Infections

ABSTRACT Decolonization measures, including mupirocin and chlorhexidine, are often prescribed to prevent Staphylococcus aureus skin and soft tissue infections (SSTI). The objective of this study was to determine the prevalence of high-level mupirocin and chlorhexidine resistance in S. aureus strains recovered from patients with SSTI before and after mupirocin and chlorhexidine administration and to determine whether carriage of a mupirocin- or chlorhexidine-resistant strain at baseline precluded S. aureus eradication. We recruited 1,089 patients with community-onset SSTI with or without S. aureus colonization. In addition to routine care, 483 patients were enrolled in a decolonization trial: 408 received intranasal mupirocin (with or without antimicrobial baths), and 258 performed chlorhexidine body washes. Patients were followed for up to 12 months with repeat colonization cultures. All S. aureus isolates were tested for high-level mupirocin and chlorhexidine resistance. At baseline, 23/1,089 (2.1%) patients carried a mupirocin-resistant S. aureus strain and 10/1,089 (0.9%) patients carried chlorhexidine-resistant S. aureus. Of 4 patients prescribed mupirocin, who carried a mupirocin-resistant S. aureus strain at baseline, 100% remained colonized at 1 month compared to 44% of the 324 patients without mupirocin resistance at baseline (P = 0.041). Of 2 patients prescribed chlorhexidine, who carried a chlorhexidine-resistant S. aureus strain at baseline, 50% remained colonized at 1 month compared to 48% of the 209 patients without chlorhexidine resistance at baseline (P = 1.0). The overall prevalence of mupirocin and chlorhexidine resistance is low in S. aureus isolates recovered from outpatients, but eradication efforts were less successful in patients carrying a mupirocin-resistant S. aureus strain at baseline.

Effectiveness of Measures to Eradicate Staphylococcus aureus Carriage in Patients with Community-Associated Skin and Soft Tissue Infections: A Randomized Trial

BACKGROUND Despite a paucity of evidence, decolonization measures are prescribed for outpatients with recurrent Staphylococcus aureus skin and soft-tissue infection (SSTI). OBJECTIVE Compare the effectiveness of 4 regimens for eradicating S. aureus carriage. DESIGN Open-label, randomized controlled trial. Colonization status and recurrent SSTI were ascertained at 1 and 4 months. SETTING Barnes-Jewish and St. Louis Childrens Hospitals, St. Louis, Missouri, 2007-2009. PARTICIPANTS Three hundred patients with community-onset SSTI and S. aureus colonization in the nares, axilla, or inguinal folds. INTERVENTIONS Participants were randomized to receive no therapeutic intervention (control subjects) or one of three 5-day regimens: 2% mupirocin ointment applied to the nares twice daily, intranasal mupirocin plus daily 4% chlorhexidine body washes, or intranasal mupirocin plus daily dilute bleach water baths. RESULTS Among 244 participants with 1-month colonization data, modified intention-to-treat analysis revealed S. aureus eradication in 38% of participants in the education only (control) group, 56% of those in the mupirocin group (P = .03 vs controls), 55% of those in the mupirocin and chlorhexidine group (P = .05), and 63% off those in the mupirocin and bleach group (P = .006). Of 229 participants with 4-month colonization data, eradication rates were 48% in the control group, 56% in the mupirocin only group (P = .40 vs controls), 54% in the mupirocin and chlorhexidine group (P = .51), and 71% in the mupirocin and bleach group (P = .02). At 1 and 4 months, recurrent SSTIs were reported by 20% and 36% of participants, respectively. CONCLUSIONS An inexpensive regimen of dilute bleach baths, intranasal mupirocin, and hygiene education effectively eradicated S. aureus over a 4-month period. High rates of recurrent SSTI suggest that factors other than endogenous colonization are important determinants of infection. Trial registration. ClinicalTrials.gov identifier: NCT00513799.

It's Not Easy Being Green: the Viridans Group Streptococci, with a Focus on Pediatric Clinical Manifestations

ABSTRACT The viridans group streptococci (VGS) are a heterogeneous group of organisms that can be human commensals, colonizing the gastrointestinal and genitourinary tracts in addition to the oral mucosa. VGS are generally considered to be of low pathogenic potential in immunocompetent individuals. However, in certain patient populations, VGS can cause invasive disease, such as endocarditis, intra-abdominal infection, and shock. Within the VGS, the rates and patterns of antimicrobial resistance vary greatly depending upon the species identification and the patient population. In general, Streptococcus mitis group organisms are resistant to more antimicrobial agents than the other VGS species. This review addresses current VGS taxonomy, in addition to the current methodologies being used in clinical microbiology laboratories for identification of VGS. Automated systems struggle overall with species level identification and susceptibility testing for VGS. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) identification is emerging as a potential alternative for organism identification. A review of recent pediatric-specific data regarding the clinical manifestations of VGS revealed that the Streptococcus anginosus group (SAG) organisms may be important pathogens in pediatric patients and that the VGS may contribute to disease in patients with cystic fibrosis. It also appears that rates of antimicrobial resistance in VGS in pediatric patients are surpassing those of the adult population.

Prevalence and Risk Factors for Asymptomatic Clostridium difficile Carriage

BACKGROUND Clostridium difficile infection (CDI) incidence has increased dramatically over the last decade. Recent studies suggest that asymptomatic carriers may be an important reservoir of C. difficile in healthcare settings. We sought to identify the prevalence and risk factors for asymptomatic C. difficile carriage on admission to the hospital. METHODS Patients admitted to Barnes-Jewish Hospital without diarrhea were enrolled from June 2010 through October 2011. Demographic information and healthcare and medication exposures 90 days prior to admission were collected. Stool specimens or rectal swabs were collected within 48 hours of admission and stored at -30°C until cultured. Clostridium difficile isolates were typed and compared with isolates from patients with CDI. RESULTS A stool/swab specimen was obtained for 259 enrolled subjects on admission. Two hundred four (79%) were not colonized, 40 (15%) had toxigenic C. difficile (TCD), and 15 (6%) had nontoxigenic C. difficile. There were no differences between TCD-colonized and -uncolonized subjects for age (mean, 56 vs 58 years; P = .46), comorbidities, admission from another healthcare facility (33% vs 24%; P = .23), or recent hospitalization (50% vs 50%; P = .43). There were no differences in antimicrobial exposures in the 90 days prior to admission (55% vs 56%; P = .91). Asymptomatic carriers were colonized with strains similar to strains from patients with CDI, but the relative proportions were different. CONCLUSIONS There was a high prevalence of TCD colonization on admission. In contrast to past studies, TCD colonization was not associated with recent antimicrobial or healthcare exposures. Additional investigation is needed to determine the role of asymptomatic TCD carriers on hospital-onset CDI incidence.

Vertically transmitted faecal IgA levels determine extra-chromosomal phenotypic variation

The proliferation of genetically modified mouse models has exposed phenotypic variation between investigators and institutions that has been challenging to control. In many cases, the microbiota is the presumed cause of the variation. Current solutions to account for phenotypic variability include littermate and maternal controls or defined microbial consortia in gnotobiotic mice. In conventionally raised mice, the microbiome is transmitted from the dam. Here we show that microbially driven dichotomous faecal immunoglobulin-A (IgA) levels in wild-type mice within the same facility mimic the effects of chromosomal mutations. We observe in multiple facilities that vertically transmissible bacteria in IgA-low mice dominantly lower faecal IgA levels in IgA-high mice after co-housing or faecal transplantation. In response to injury, IgA-low mice show increased damage that is transferable by faecal transplantation and driven by faecal IgA differences. We find that bacteria from IgA-low mice degrade the secretory component of secretory IgA as well as IgA itself. These data indicate that phenotypic comparisons between mice must take into account the non-chromosomal hereditary variation between different breeders. We propose faecal IgA as one marker of microbial variability and conclude that co-housing and/or faecal transplantation enables analysis of progeny from different dams.

Multicenter Evaluation of the Vitek MS Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry System for Identification of Gram-Positive Aerobic Bacteria

ABSTRACT Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF) is gaining momentum as a tool for bacterial identification in the clinical microbiology laboratory. Compared with conventional methods, this technology can more readily and conveniently identify a wide range of organisms. Here, we report the findings from a multicenter study to evaluate the Vitek MS v2.0 system (bioMérieux, Inc.) for the identification of aerobic Gram-positive bacteria. A total of 1,146 unique isolates, representing 13 genera and 42 species, were analyzed, and results were compared to those obtained by nucleic acid sequence-based identification as the reference method. For 1,063 of 1,146 isolates (92.8%), the Vitek MS provided a single identification that was accurate to the species level. For an additional 31 isolates (2.7%), multiple possible identifications were provided, all correct at the genus level. Mixed-genus or single-choice incorrect identifications were provided for 18 isolates (1.6%). Although no identification was obtained for 33 isolates (2.9%), there was no specific bacterial species for which the Vitek MS consistently failed to provide identification. In a subset of 463 isolates representing commonly encountered important pathogens, 95% were accurately identified to the species level and there were no misidentifications. Also, in all but one instance, the Vitek MS correctly differentiated Streptococcus pneumoniae from other viridans group streptococci. The findings demonstrate that the Vitek MS system is highly accurate for the identification of Gram-positive aerobic bacteria in the clinical laboratory setting.

Optimizing Identification of Clinically Relevant Gram-Positive Organisms by Use of the Bruker Biotyper Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry System

ABSTRACT Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) can be used as a method for the rapid identification of microorganisms. This study evaluated the Bruker Biotyper (MALDI-TOF MS) system for the identification of clinically relevant Gram-positive organisms. We tested 239 aerobic Gram-positive organisms isolated from clinical specimens. We evaluated 4 direct-smear methods, including “heavy” (H) and “light” (L) smears, with and without a 1-μl direct formic acid (FA) overlay. The quality measure assigned to a MALDI-TOF MS identification is a numerical value or “score.” We found that a heavy smear with a formic acid overlay (H+FA) produced optimal MALDI-TOF MS identification scores and the highest percentage of correctly identified organisms. Using a score of ≥2.0, we identified 183 of the 239 isolates (76.6%) to the genus level, and of the 181 isolates resolved to the species level, 141 isolates (77.9%) were correctly identified. To maximize the number of correct identifications while minimizing misidentifications, the data were analyzed using a score of ≥1.7 for genus- and species-level identification. Using this score, 220 of the 239 isolates (92.1%) were identified to the genus level, and of the 181 isolates resolved to the species level, 167 isolates (92.2%) could be assigned an accurate species identification. We also evaluated a subset of isolates for preanalytic factors that might influence MALDI-TOF MS identification. Frequent subcultures increased the number of unidentified isolates. Incubation temperatures and subcultures of the media did not alter the rate of identification. These data define the ideal bacterial preparation, identification score, and medium conditions for optimal identification of Gram-positive bacteria by use of MALDI-TOF MS.

Interleukin-7 Ameliorates Immune Dysfunction and Improves Survival in a 2-Hit Model of Fungal Sepsis

BACKGROUND Secondary hospital-acquired fungal infections are common in critically-ill patients and mortality remains high despite antimicrobial therapy. Interleukin-7 (IL-7) is a potent immunotherapeutic agent that improves host immunity and has shown efficacy in bacterial and viral models of infection. This study examined the ability of IL-7, which is currently in multiple clinical trials (including hepatitis and human immunodeficiency virus), to improve survival in a clinically relevant 2-hit model of fungal sepsis. METHODS Mice underwent cecal ligation and puncture to induce peritonitis. Four days later, surviving mice had intravenous injection with Candida albicans. Following Candida infection, mice were treated with IL-7 or saline control. The effect of IL-7 on host immunity and survival was recorded. RESULTS IL-7 ameliorated the loss of immune effector cells and increased lymphocyte functions, including activation, proliferation, expression of adhesion molecules, and interferon-γ production. These beneficial effects of IL-7 were associated with an increase in global immunity as reflected by an enhanced delayed type hypersensitivity response and a 1.7-fold improvement in survival. CONCLUSIONS The present findings showing that IL-7 improves survival in fungal sepsis, together with its previously reported efficacy in bacterial and viral infectious models, further supports its use as a novel immunotherapeutic in sepsis.

Developmental dynamics of the preterm infant gut microbiota and antibiotic resistome

Development of the preterm infant gut microbiota is emerging as a critical research priority1. Since preterm infants almost universally receive early and often extended antibiotic therapy2, it is important to understand how these interventions alter gut microbiota development3–6. Analysis of 401 stools from 84 longitudinally sampled preterm infants demonstrates that meropenem, cefotaxime and ticarcillin–clavulanate are associated with significantly reduced species richness. In contrast, vancomycin and gentamicin, the antibiotics most commonly administered to preterm infants, have non-uniform effects on species richness, but these can be predicted with 85% accuracy based on the relative abundance of only two bacterial species and two antibiotic resistance (AR) genes at treatment initiation. To investigate resistome development, we functionally selected resistance to 16 antibiotics from 21 faecal metagenomic expression libraries. Of the 794 AR genes identified, 79% had not previously been classified as AR genes. Combined with deep shotgun sequencing of all stools, we find that multidrug-resistant members of the genera Escherichia, Klebsiella and Enterobacter, genera commonly associated with nosocomial infections, dominate the preterm infant gut microbiota. AR genes that are enriched following specific antibiotic treatments are generally unique to the specific treatment and are highly correlated with the abundance of a single species. The most notable exceptions include ticarcillin–clavulanate and ampicillin, both of which enrich for a large number of overlapping AR genes, and are correlated with Klebsiella pneumoniae. We find that all antibiotic treatments are associated with widespread collateral microbiome impact by enrichment of AR genes that have no known activity against the specific antibiotic driver.