Enzler, Mark J.

ABSTRACT An inherent problem in the diagnostic PCR assay is the presence of ill-defined inhibitors of amplification which may cause false-negative results. Addition of an amplifiable fragment of foreign DNA in the PCR to serve as a hybrid internal control (HIC) would allow for a simple way to identify specimens containing inhibitors. Two oligonucleotide hybrid primers were synthesized to contain nucleic acid sequences of the Chlamydia pneumoniae 16S rRNA primers in a position flanking two primers that target the sequences of a 650-bp lambda phage DNA segment. By using the hybrid primers, hybrid DNA comprising a large sequence of lambda phage DNA flanked by short pieces of chlamydia DNA was subsequently generated by PCR, cloned into a plasmid vector, and purified. Plasmids containing the hybrid DNA were diluted and used as a HIC by adding them to each C. pneumoniae PCR test. Consequently, C. pneumoniae primers were able to amplify both chlamydia DNA and the HIC DNA. The production of a 689-bp HIC DNA band on an acrylamide gel indicated that the specimen contained no inhibitors and that internal conditions were compatible with PCR. Subsequently, a biotinylated RNA probe for the HIC was transcribed from a nested sequence of the HIC and was used for its hybridization. Detection of the HIC DNA-RNA hybrid was achieved by enzyme immunoassay (EIA). This PCR-EIA system with a HIC was initially tested with 12 previously PCR-positive and 14 previously PCR-negative specimens. Of the 12 PCR-positive specimens, 11 were reconfirmed as positive; 1 had a negative HIC value, indicating inhibition. Of the 14 previously PCR-negative specimens, 13 were confirmed as true negative; 1 had a negative HIC value, indicating inhibition. The assay was then used with 237 nasopharyngeal specimens from patients with pneumonia. Twenty-one of 237 (8.9%) were positive for C. pneumoniae , and 42 (17.7%) were found to inhibit the PCR. Specimens showing inhibitory activity were diluted 1:10 and were retested. Ten specimens were still inhibitory to the PCR and required further DNA purification. No additional positive samples were detected and 3 nasopharyngeal specimens remained inhibitory to PCR. Coamplification of a HIC DNA can help confirm true-negative PCR results by ruling out the presence of inhibitors of DNA amplification.

Abstract Background Tumor necrosis factor (TNF)-α antagonist therapy has revolutionized the practice of management of inflammatory bowel disease (IBD); however, these medications carry a boxed warning from the Food and Drug Administration for risk of serious infection. We aimed to study the invasive fungal infection, histoplasmosis, in the setting of TNF-α antagonist therapy. Methods We performed a retrospective review of patients with IBD receiving TNF-α antagonist therapy who developed histoplasmosis during the time period January 2001–May 2018 at the Mayo Clinic, Rochester, MN. The medical records of patients were reviewed for demographics, medications, symptoms, diagnosis, treatment, and outcomes including mortality. IBD was diagnosed by biopsy, radiographic, or endoscopic evidence of disease. Results We identified 49 patients (age range 19–74; median 44 years) with a confirmed diagnosis of histoplasmosis while receiving a TNF-α antagonist. 73.5% of cases were classified as disseminated. Median time from starting TNF-α antagonist to histoplasmosis diagnosis was 2.1 years. Liposomal amphotericin B was given in 17 cases as the initial treatment. Itraconazole was given to all 49 patients. Initial treatment was split evenly between inpatient (49%) and outpatient (51%) locations with 6 patients (12%) requiring ICU-level care. Median length of stay was 9.5 days. The total length of treatment for all antifungals was 38.4 weeks, with 20.4% of patients developing documented antifungal side effects. TNF-α antagonist was continued in 9 patients (18.4%) and another 10 patients resumed TNF-α antagonist. Half of those who resumed TNF-α antagonists were on antifungal therapy. There was one histoplasmosis recurrence while off TNF-α antagonist, and three deaths (6%). Conclusion Histoplasmosis outcomes in IBD patients on TNF-α antagonists were mostly favorable; however, approximately half required hospitalization. Many patients were young with few co-morbidities, and over one-third were able to continue or resume TNF-α antagonists without documented recurrence of histoplasmosis. Practitioners should be vigilant for histoplasmosis infections in this patient population who reside in histoplasma-endemic regions. Disclosures All authors: No reported disclosures.

SummaryErysipelothrix rhusiopathiae is a facultatively anaerobic Gram‐positive bacillus found mostly in swine, fish and sheep. E. rhusiopathiae classically causes cutaneous eruptions in butchers, fish handlers and veterinarians. Based solely on case reports, 90% of E. rhusiopathiae bloodstream infections (BSI) have been associated with infective endocarditis (IE). To assess the true frequency of IE in E. rhusiopathiae BSI as well as other clinical associations, we performed a retrospective cohort analysis of E. rhusiopathiae BSI at Mayo Clinic. This is a single‐centre, retrospective study conducted between 1/1/1994 and 20/6/2016 at Mayo Clinic in Rochester, MN. Medical records were reviewed for demographics, E. rhusiopathiae BSI, anti‐microbial susceptibilities, incidence of IE, patient comorbidities, intensive care unit (ICU) admission and duration of antibiotics. Five cases of E. rhusiopathiae BSI were identified. Risk factors included animal exposures, immunosuppression, diabetes and kidney disease. All cases involved penicillin‐sensitive strains and high‐grade BSI. Four cases showed no signs of IE on transesophageal echocardiogram. All patients recovered fully with intravenous antibiotics. Our retrospective review illustrates that E. rhusiopathiae can cause invasive BSI in the absence of IE and that the previously reported 90% association between BSI and IE may be overestimated due to reporting bias. E. rhusiopathiae should be suspected in any patient with Gram‐positive bacilli in blood cultures and the aforementioned risk factors. A limitation of our study was the low sample size, and future studies may involve multicentre collaborations and the use of polymerase chain reaction (PCR) or serologic testing to increase the number of diagnoses..

AbstractBackground. The long-term outcome of patients with pyogenic vertebral osteomyelitis (PVO) has not been fully assessed.Methods. We conducted a retrospective cohort study to describe the long-term outcome of PVO and to assess risk factors for treatment failure in patients evaluated at our institution between 1994 and 2002. Patients were observed until July 1, 2013.Results. Two hundred sixty patients with PVO were included in this study. Twenty-seven percent (70) of patients developed their infection after an invasive spinal procedure. Staphylococcus aureus accounted for 40% (103) of infections. Forty-nine percent (128) of patients underwent spinal surgery as part of their initial therapy. The median duration of parenteral antimicrobial therapy was 42 days (interquartile range, 38–53). The estimated 2-, 5-, and 10-year cumulative probability of treatment failure-free survival was 72%, 69%, and 69%, respectively. Seventy-five percent of patients who developed treatment failure did so within 4.7 months of diagnosis. Residual neurological defects and persistent back pain were seen in 16% and 32% of patients, respectively. In a multivariate analysis, longer duration of symptoms before diagnosis and having an infection with S. aureus were associated with increased risk of treatment failure.Conclusions. Increasing duration of symptoms and infection with S. aureus were associated with treatment failure in patients with PVO. Most treatment failures occurred early after initiation of treatment. Pyogenic vertebral osteomyelitis is associated with a high 2-year failure rate. Persistent neurological deficits and back pain are common after therapy.

Background: Because both daptomycin and statins may increase creatine phosphokinase (CPK) levels, the manufacturer of daptomycin suggests considering holding statins during daptomycin therapy. Published evidence suggests potential detrimental effects of withdrawing statin therapy. Objectives: The objectives of this study were to determine the impact of concurrent statin therapy on peak CPK values, incidence of CPK elevation in patients receiving daptomycin therapy, and clinical factors associated with increased risk of developing CPK elevation. Methods: This was a single-center, retrospective cohort study of patients ≥18 years of age who received daptomycin for ≥72 hours and had ≥1 follow-up CPK during a 5-year period. A Kaplan-Meier curve was used to evaluate time to CPK elevation. Cox regression analyses were used to compare the risk of developing elevated CPK between 3 study groups: those receiving daptomycin alone, daptomycin with concurrent statin therapy, and statin therapy held while on daptomycin. Results: 498 patients were included in the study—384 received daptomycin alone, 63 received daptomycin concurrent with statin, and 51 with statin held during daptomycin therapy. Cumulative incidence of CPK elevation was 5.1% and 12% at 7 and 14 days. Those on daptomycin and statin concurrent therapy demonstrated an approximately 2-fold risk of CPK elevation compared with those having their statin therapy held, but the overall group effect was not statistically significant ( P = .17). Conclusions: Our findings suggest that holding statin during daptomycin therapy may not be necessary, but may indicate need for increased frequency of CPK monitoring when these medications are used concurrently.