Kristin Fiebelkorn

Practical Disk Diffusion Method for Detection of Inducible Clindamycin Resistance in Staphylococcus aureus and Coagulase-Negative Staphylococci

ABSTRACT Resistance to macrolides in staphylococci may be due to active efflux (encoded by msrA) or ribosomal target modification (macrolide-lincosamide-streptogramin B [MLSB] resistance; usually encoded by ermA or ermC). MLSB resistance is either constitutive or inducible following exposure to a macrolide. Induction tests utilize closely approximated erythromycin and clindamycin disks; the flattening of the clindamycin zone adjacent to the erythromycin disk indicates inducible MLSB resistance. The present study reassessed the reliability of placing erythromycin and clindamycin disks in adjacent positions (26 to 28 mm apart) in a standard disk dispenser, compared to distances of 15 or 20 mm. A group of 130 clinical isolates of Staphylococcus aureus and 100 isolates of erythromycin-resistant coagulase-negative staphylococci (CNS) were examined by disk approximation; all CNS isolates and a subset of S. aureus isolates were examined by PCR for ermA, ermC, and msrA. Of 114 erythromycin-resistant S. aureus isolates, 39 demonstrated constitutive resistance to clindamycin, while 33 showed inducible resistance by disk approximation at all three distances. Only one isolate failed to clearly demonstrate induction at 26 mm. Of 82 erythromycin-resistant CNS isolates that contained ermA or ermC, 57 demonstrated constitutive clindamycin resistance, and 25 demonstrated inducible resistance, at 20 and 26 mm. None of the 42 S. aureus isolates or 18 CNS isolates containing only msrA and none of the erythromycin-susceptible isolates yielded positive disk approximation tests. Simple placement of erythromycin and clindamycin disks at a distance achieved with a standard disk dispenser allowed detection of 97% of S. aureus strains and 100% of CNS strains with inducible MLSB resistance in this study.

Clinical Evaluation of Two Methods for Genotyping Hepatitis C Virus Based on Analysis of the 5′ Noncoding Region

ABSTRACT We compared the performance characteristics of a standardized direct sequencing method (TRUGENE HCV 5′NC; Visible Genetics Inc., Toronto, Ontario, Canada) and a reverse hybridization line probe assay (INNO-LiPA HCV II; Bayer Corp., Tarrytown, N.Y.) for genotyping of hepatitis C virus (HCV). Both methods are based on detection of sequence heterogeneity in the 5′ noncoding (5′NC) region. Concordance between the genotyping methods was assessed by testing 172 samples representing the six major genotypes. Sequence analysis of the more phylogenetically informative nonstructural 5B (NS5B) region was also done with 148 (86%) samples to confirm the accuracy of and resolve discrepancies between the 5′NC genotyping results. The sensitivities of the methods were assessed by using the 5′NC amplicon from both the qualitative and quantitative AMPLICOR HCV tests (Roche Diagnostics Corp., Indianapolis, Ind.). The ability of the methods to detect mixed-genotype infections was determined with mixtures of two different genotypes at relative concentrations ranging from 1 to 50%. Both 5′NC methods were able to genotype 99.4% of the samples with type agreement for 99.5% and subtype agreement for 68.2% of the samples. No or ambiguous subtype results were found by the line probe assay for 16.5% and by the TRUGENE 5′NC test for 17.1% of the samples. Discrepancies occurred between the line probe assay and NS5B results at the type level for 1.4% of the samples and at the subtype level for 14.2% of the samples. Discrepancies also occurred between the TRUGENE 5′NC and NS5B results at the type level for 2% of the samples and at the subtype level for 8.1% of the samples. We also found two distinct strains of HCV classified as type 2 by analysis of the 5′NC region that were type 1 by analysis of the NS5B region. The sensitivities of the two 5′NC genotyping methods were comparable and dependent on the amplification test used (∼103 IU/ml with the qualitative HCV RNA tests and ∼105 IU/ml with the quantitative HCV RNA tests). Genotype mixtures were successfully identified at a relative concentration of 5% by the line probe assay and 10% by the TRUGENE 5′NC test. In conclusion, the performance characteristics of the 5′NC methods were similar and both methods produced accurate results at the genotype level but neither method should be used for subtyping.

Detection of Inducible Clindamycin Resistance of Staphylococci in Conjunction with Performance of Automated Broth Susceptibility Testing

ABSTRACT This study has shown that inducible clindamycin resistance in staphylococci can be detected by disk testing on sheep blood agar inoculum purity plates used with the bioMerieux VITEK 2. Tests of 150 erythromycin-resistant isolates correlated with standard D-zone tests on Mueller-Hinton agar and with PCR for erm(A), erm(C), and msr(A).

Timing of Specimen Collection for Blood Cultures from Febrile Patients with Bacteremia

ABSTRACT Bloodstream infections are an important cause of morbidity and mortality. Physician orders for blood cultures often specify that blood specimens be collected at or around the time of a temperature elevation, presumably as a means of enhancing the likelihood of detecting significant bacteremia. In a multicenter study, which utilized retrospective patient chart reviews as a means of collecting data, we evaluated the timing of blood culture collection in relation to temperature elevations in 1,436 patients with bacteremia and fungemia. The likelihood of documenting bloodstream infections was not significantly enhanced by collecting blood specimens for culture at the time that patients experienced temperature spikes. A subset analysis based on patient age, gender, white blood cell count and specific cause of bacteremia generally also failed to reveal any associations.

Susceptibility of Neisseria meningitidis to 16 Antimicrobial Agents and Characterization of Resistance Mechanisms Affecting Some Agents

ABSTRACT Neisseria meningitidis represents a pathogen of great public health importance in both developed and developing countries. Resistance to some antimicrobial agents used either for therapy of invasive infections or for prophylaxis of case contacts has long been recognized, although specific guidelines for susceptibility testing have not been fully developed. We have examined the susceptibilities of a collection of 442 meningococcal clinical isolates from 15 countries to 16 antimicrobial agents. These included isolates recovered between 1917 and 2004, with representatives of all major serogroups. All isolates were tested by the Clinical and Laboratory Standards Institute (formerly NCCLS) broth microdilution method using Mueller-Hinton lysed horse blood broth, while a subset of 102 isolates was tested by agar dilution using Mueller-Hinton sheep blood agar. Most isolates provided adequate growth for MIC determinations by both broth and agar methods. Growth in broth was enhanced by CO2 incubation and was required for two strains (1.7%). MICs of the study drugs compared favorably between the broth and agar methods (79 to 100% essential agreement), and MICs also generally agreed closely (92 to 100% essential agreement, excluding azithromycin) between broth tests incubated in the two different atmospheres. Elevated penicillin and ampicillin MICs (≥0.12 μg/ml and ≥0.25 μg/ml, respectively) occurred in 14.3% and 8.6% of strains and were associated with polymorphisms of the penA gene encoding a modified penicillin-binding protein 2. None of the 442 isolates produced beta-lactamase. Elevated tetracycline and doxycycline (but not minocycline) MICs were associated with efflux-mediated resistance encoded by tet(B) in 13 strains. Resistance to sulfisoxazole in 21.7% of strains and to trimethoprim-sulfamethoxazole in 21.0% resulted from polymorphisms of folP encoding a modified dihydropteroate synthetase. Seven strains were resistant to rifampin due to mutations in the rpoB gene, and two strains were resistant to chloramphenicol due to production of chloramphenicol acetyltransferase mediated by catP. Two strains had reduced quinolone susceptibility due to mutations of gyrA. The determination of the susceptibilities of a large group of meningococcal strains (including strains with characterized resistance mechanisms) to 16 antimicrobial agents has served as the essential first step in defining susceptibility testing breakpoints specific for this organism.

High priority for hepatitis C screening in safety net hospitals: Results from a prospective cohort of 4582 hospitalized baby boomers

Low‐income populations are disproportionately affected by hepatitis C virus (HCV) infection. Thus, implementing baby boomer screening (born 1945‐1965) for HCV may be a high priority for safety net hospitals. We report the prevalence and predictors of HCV infection and advanced fibrosis or cirrhosis based on the Fibrosis‐4 score plus imaging for a baby boomer cohort admitted to a safety net hospital over a 21‐month interval with >9 months of follow‐up. Anti‐HCV antibody testing was performed for 4582, or 90%, of all never‐screened patients, of whom 312 (6.7%) tested positive. Adjusted odds ratios of testing anti‐HCV‐positive were 2.66 for men versus women (P < 0.001), 1.25 for uninsured versus insured (P = 0.06), 0.70 for Hispanics versus non‐Hispanic whites (P = 0.005), and 0.93 per year of age (P < 0.001). Among 287 patients tested for HCV RNA (91% of all anti‐HCV‐positive cases), 175 (61%) were viremic (3.8% overall prevalence in cohort), which was 5% less likely per year of age (P < 0.03). Noninvasive staging of 148 (84.6%) chronic HCV patients identified advanced fibrosis or cirrhosis in 50 (33.8%), with higher adjusted odds ratios of 3.21 for Hispanics versus non‐Hispanic whites/Asians (P = 0.02) and 1.18 per year of age (P = 0.001). Other factors associated with significantly higher adjusted odds ratios of advanced fibrosis or cirrhosis were alcohol abuse/dependence, obesity, and being uninsured. Conclusion: In this low‐income, hospitalized cohort, 4% of 4582 screened baby boomers were diagnosed with chronic HCV, nearly twice the rate in the community; one‐third had noninvasive testing that indicated advanced fibrosis or cirrhosis, which was significantly more likely for Hispanics, those of older age, those with obesity, those with alcohol abuse/dependence, and those who lacked insurance. (Hepatology 2015;62:1388–1395)

Mutations in folP Associated with Elevated Sulfonamide MICs for Neisseria meningitidis Clinical Isolates from Five Continents

ABSTRACT Sulfonamide resistance in meningococci is associated with mutations in the chromosomal gene folP, which encodes dihydropteroate synthase. Several mutations associated with resistance have been previously described, including amino acid substitutions at codons 31 and 194, a glycine-serine insertion at codons 195 and 196, and, recently, an additional mutation at nucleotide 682 (C682A). In this study, sulfisoxazole MICs were determined for 424 geographically diverse clinical isolates of Neisseria meningitidis, including all major subtypes. A subset of 134 isolates with MICs ranging from 0.5 to >64 μg/ml were assayed for the C682A mutation by real-time PCR, and 25 isolates were selected for folP gene sequencing. All isolates for which the sulfisoxazole MIC was ≥8 possessed the C682A mutation by real-time PCR or folP sequencing, and 34 of 35 isolates with a MIC of ≤2 lacked this mutation. Of 16 sequenced isolates for which the sulfisoxazole MIC was ≥4, 15 possessed previously described mutations, including 10 at codon 31, 1 at codon 194, and 4 with the 2-amino-acid insertion codons 195 and 196; all 16 possessed the C682A mutation. The C682A mutation predicted elevated sulfonamides MICs for a large number of geographically diverse clinical isolates of meningococci. Detection of this mutation by real-time PCR or other methods may allow more wide-scale detection of meningococcal isolates with for which the sulfonamide MICs are elevated without resorting to multiple assays or folP gene sequencing, providing a simple, high-throughput screening method for use in public health and epidemiologic settings.

Implementing hospital-based baby boomer hepatitis c virus screening and linkage to care: Strategies, results, and costs

BACKGROUND/OBJECTIVE The US Preventive Services Task Force recommends 1-time hepatitis C virus (HCV) screening of all baby boomers (born 1945-1965). However, little is known about optimal ways to implement HCV screening, counseling, and linkage to care. We developed strategies following approaches used for HIV to implement baby boomer HCV screening in a hospital setting and report results as well as costs. DESIGN/PATIENTS Prospective cohort of 6140 baby boomers admitted to a safety-net hospital in South Texas from December 1, 2012 to January 31, 2014 and followed to December 10, 2014. PROCEDURES/MEASUREMENTS The HCV screening program included clinician/staff education, electronic medical record algorithm for eligibility and order entry, opt-out consent, anti-HCV antibody test with reflex HCV RNA, personalized inpatient counseling, and outpatient case management. Outcomes were anti-HCV antibody-positive and HCV RNA-positive results. RESULTS Of 3168 eligible patients, 240 (7.6%) were anti-HCV positive, which was more likely (P < 0.05) for younger age, men, and uninsured. Of 214 (89.2%) patients tested for HCV RNA, 134 (4.2% of all screened) were positive (chronic HCV). Among patients with chronic HCV, 129 (96.3%) were counseled, 108 (80.6%) received follow-up primary care, and 52 (38.8%) received hepatology care. Five patients initiated anti-HCV therapy. Total costs for start-up and implementation for 14 months were

High priority for hepatitis C screening in safety net hospitals

286,482. CONCLUSIONS This inpatient HCV screening program diagnosed chronic HCV infection in 4.2% of tested patients and linked >80% to follow-up care. Yet access to therapy is challenging for largely uninsured populations, and most programmatic costs of the program are not currently covered.

Development and Verification of an Automated Sample Processing Protocol for Quantitation of Human Immunodeficiency Virus Type 1 RNA in Plasma

Low‐income populations are disproportionately affected by hepatitis C virus (HCV) infection. Thus, implementing baby boomer screening (born 1945‐1965) for HCV may be a high priority for safety net hospitals. We report the prevalence and predictors of HCV infection and advanced fibrosis or cirrhosis based on the Fibrosis‐4 score plus imaging for a baby boomer cohort admitted to a safety net hospital over a 21‐month interval with >9 months of follow‐up. Anti‐HCV antibody testing was performed for 4582, or 90%, of all never‐screened patients, of whom 312 (6.7%) tested positive. Adjusted odds ratios of testing anti‐HCV‐positive were 2.66 for men versus women (P < 0.001), 1.25 for uninsured versus insured (P = 0.06), 0.70 for Hispanics versus non‐Hispanic whites (P = 0.005), and 0.93 per year of age (P < 0.001). Among 287 patients tested for HCV RNA (91% of all anti‐HCV‐positive cases), 175 (61%) were viremic (3.8% overall prevalence in cohort), which was 5% less likely per year of age (P < 0.03). Noninvasive staging of 148 (84.6%) chronic HCV patients identified advanced fibrosis or cirrhosis in 50 (33.8%), with higher adjusted odds ratios of 3.21 for Hispanics versus non‐Hispanic whites/Asians (P = 0.02) and 1.18 per year of age (P = 0.001). Other factors associated with significantly higher adjusted odds ratios of advanced fibrosis or cirrhosis were alcohol abuse/dependence, obesity, and being uninsured. Conclusion: In this low‐income, hospitalized cohort, 4% of 4582 screened baby boomers were diagnosed with chronic HCV, nearly twice the rate in the community; one‐third had noninvasive testing that indicated advanced fibrosis or cirrhosis, which was significantly more likely for Hispanics, those of older age, those with obesity, those with alcohol abuse/dependence, and those who lacked insurance. (Hepatology 2015;62:1388–1395)