Donald Jungkind

The inhibition of Staphylococcus epidermidis biofilm formation by vancomycin-modified titanium alloy and implications for the treatment of periprosthetic infection.

Peri-prosthetic infections are notoriously difficult to treat as the biomaterial implant is ideal for bacterial adhesion and biofilm formation, resulting in decreased antibiotic sensitivity. Previously, we reported that vancomycin covalently attached to a Ti alloy surface (Vanc-Ti) could prevent bacterial colonization. Herein we examine the effect of this Vanc-Ti surface on Staphylococci epidermidis, a Gram-positive organism prevalent in orthopaedic infections. By direct colony counting and fluorescent visualization of live bacteria, S. epidermidis colonization was significantly inhibited on Vanc-Ti implants. In contrast, the gram-negative organism Escherichia coli readily colonized the Vanc-Ti rod, suggesting retention of antibiotic specificity. By histochemical and SEM analysis, Vanc-Ti prevented S. epidermidis biofilm formation, even in the presence of serum. Furthermore, when challenged multiple times with S. epidermidis, Vanc-Ti rods resisted bacterial colonization. Finally, when S. epidermidis was continuously cultured in the presence of Vanc-Ti, the bacteria maintained a Vanc sensitivity equivalent to the parent strain. These findings indicate that antibiotic derivatization of implants can result in a surface that can resist bacterial colonization. This technology holds great promise for the prevention and treatment of periprosthetic infections.

Microbiological, clinical, and surgical features of fungal prosthetic joint infections: a multi-institutional experience.

Periprosthetic joint infection is one of the most dreaded and complex complications of total joint arthroplasty. Periprosthetic joint infection is now the major cause of failure following total knee arthroplasty1 and the third most common cause of failure following total hip arthroplasty2. It is estimated that the prevalence of periprosthetic joint infection may be on the rise3. A wide variety of pathogens are known to cause periprosthetic joint infection, with the majority of infections being caused by gram-positive bacteria, especially staphylococcal species4,5. The treatment of a confirmed periprosthetic joint infection often includes the need for surgical intervention, and two-stage exchange arthroplasty is the most common mode of surgical treatment in North America. Two-stage exchange arthroplasty relies on removal of all foreign material and insertion of an antibiotic-impregnated cement spacer for the purpose of delivering high doses of antibiotics locally in the interval of time between the resection arthroplasty and subsequent reimplantation. Periprosthetic infection with fungi, although rare, represents a diagnostic and therapeutic challenge to which clear guidelines have not yet been established. It is not known if the protocol for treatment of a bacterial periprosthetic joint infection can also be applied in the same manner to fungal infections. Patients with fungal periprosthetic joint infection are believed to be a different type of host with decreased cellular immunity, mostly due to an underlying cause of immunosuppression, such as malignant disease, drug therapies (antineoplastic agents, corticosteroids, or immunosuppressive drugs), overuse or inappropriate use of antibiotics, and indwelling catheters (urinary or parenteral hyperalimentation). Other factors, such as diabetes, tuberculosis, intravenous drug use, and acquired immunosuppressive disease, are associated with an increased frequency of mycotic infection6. The lack of reliable antifungal medications for systemic and, in particular, local delivery poses a real challenge …

Automation of laboratory testing for infectious diseases using the polymerase chain reaction — our past, our present, our future

While it is an extremely powerful and versatile assay method, polymerase chain reaction (PCR) can be a labor-intensive process. Since the advent of commercial test kits from Roche and the semi-automated microwell Amplicor system, PCR has become an increasingly useful and widespread clinical tool. However, more widespread acceptance of molecular testing will depend upon automation that allows molecular assays to enter the routine clinical laboratory. The forces driving the need for automated PCR are the requirements for diagnosis and treatment of chronic viral diseases, economic pressures to develop more automated and less expensive test procedures similar to those in the clinical chemistry laboratories, and a shortage in many areas of qualified laboratory personnel trained in the types of manual procedures used in past decades. The automated Roche COBAS AMPLICOR system has automated the amplification and detection process. Specimen preparation remains the most labor-intensive part of the PCR testing process, accounting for the majority of the hands-on-time in most of the assays. A new automated specimen preparation system, the COBAS AmpliPrep, was evaluated. The system automatically releases the target nucleic acid, captures the target with specific oligonucleotide probes, which become attached to magnetic beads via a biotin-streptavidin binding reaction. Once attached to the beads, the target is purified and concentrated automatically. Results of 298 qualitative and 57 quantitative samples representing a wide range of virus concentrations analyzed after the COBAS AmpliPrep and manual specimen preparation methods, showed that there was no significant difference in qualitative or quantitative hepatitis C virus (HCV) assay performance, respectively. The AmpliPrep instrument decreased the time required to prepare serum or plasma samples for HCV PCR to under 1 min per sample. This was a decrease of 76% compared to the manual specimen preparation method. Systems that can analyze more samples with higher throughput and that can answer more questions about the nature of the microbes that we can presently only detect and quantitate will be needed in the future.

In vitro susceptibility patterns of methicillin-resistant Staphylococcus aureus and coagulase-negative Staphylococcus corneal isolates to antibiotics.

Purpose: To determine the in vitro susceptibility of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant coagulase-negative Staphylococcus (MRCNS) isolates to various antibiotics. Methods: All cases of bacterial keratitis caused by Staphylococcus species during 2006 and 2007 were identified. The isolates were divided according to species and susceptibility to methicillin into 4 groups: methicillin-susceptible S. aureus, methicillin-susceptible coagulase-negative Staphylococcus, MRSA, and MRCNS. Routine susceptibility testing for Staphylococcus species to methicillin and 19 other antibiotics was performed using the MicroScan POS Breakpoint Combo Panel Type 20. Results: One hundred fifty-seven isolates were identified. Forty isolates were S. aureus, including 21 MRSA, and 117 isolates were coagulase-negative Staphylococcus, including 29 MRCNS. All MRSA isolates were susceptible to gentamicin, linezolid, rifampin, tetracycline, and vancomycin and were resistant to penicillin, cefazolin, cefepime, azithromycin, erythromycin, and ofloxacin. Ninety percent of MRSA isolates were resistant to fourth-generation fluoroquinolones. All MRCNS isolates were susceptible to vancomycin, chloramphenicol, linezolid, and rifampin and were resistant to penicillin, cefazolin, cefepime, and azithromycin. Sixty-five percent of the MRCNS isolates were susceptible to fourth-generation fluoroquinolones and gentamicin. Conclusions: All MRSA and MRCNS isolates were sensitive to vancomycin, linezolid, and rifampin. MRSA isolates were generally sensitive to gentamicin and tetracycline and resistant to fourth-generation fluoroquinolones. MRCNS isolates were not consistently sensitive to gentamicin, tetracycline, or fourth-generation fluoroquinolones.

Viral Burden and Disease Progression in HIV-1-Infected Patients With Sickle Cell Anemia

The spleen and lymph nodes are major sites of human immunodeficiency virus type 1 (HIV‐1) replication, mutation, and genetic variation in vivo. If a major portion of the lymphatic tissue, such as the spleen, is removed or otherwise is unavailable for invasion by the HIV‐1 virus, will the course of the infection be altered, resulting in a prolonged symptom‐free interval or even increased survival? The spleen of most adults with sickle cell anemia (SS) is nonfunctional due to recurrent episodes of microinfarction. If autosplenectomized SS patients are exposed to HIV‐1, they may be ideal candidates to examine the question of whether absence of splenic function at the time of infection will positively alter the course of HIV‐1‐related disease. All SS patients with a diagnosis of HIV‐1 infection at five university sickle cell centers were included in the patient cohort. Patients in active treatment or in follow‐up (group A, n = 11) underwent a series of quantitative viral studies to determine their HIV‐1 viral burden. The studies included the branched‐DNA signal amplification assay, quantitative DNA‐polymerase chain reaction (PCR), quantitative reverse transcription (RT)‐initiated–PCR, and in situ PCR. All patients who died of the complications of the acquired immunodeficiency syndrome (AIDS) or of SS, lost to follow‐up, or were otherwise unavailable for study (Group B: n = 7) were included in the total patient group. None of the patients in group B underwent quantitative viral studies. In addition, a control population (group C, n = 36) of HIV‐1–infected African Americans without SS, of similar age and gender to the SS patients, were compared with the study population for outcomes. In eight of 11 active patients (group A), the CD4+ T‐lymphocyte counts were normal and viral burdens were low for an average of 10.25 years following diagnosis. These eight patients all from group A were the only long‐term nonprogressors (44%) among a total of 18 SS patients (groups A and B). In group C (control), only five patients of 36 were long‐term nonprogressors (13.9%). Five patients (28%) of the total SS group (groups A and B) succumbed to AIDS. One of the five was from Group A. The evaluation of a limited number of adult individuals suggests that a significant proportion of HIV‐1–seropositive SS patients (44%) may be asymptomatic long‐term nonprogressors. In these patients, the CD4+ T‐lymphocyte counts remained high and their viral burdens were remarkably lower than in non‐SS HIV‐1–seropositive individuals. Whereas this study does not prove an “autosplenectomy” hypothesis, it suggests that in patients with both SS and HIV‐1 infection, the retroviral disease may be ameliorated by host factors of which absence of splenic function prior to HIV‐1 infection may be one. Am. J. Hematol. 59:199–207, 1998.

Systemic antimicrobial therapy in skin and skin structure infections: Comparison of temafloxacin and ciprofloxacin

Temafloxacin is an investigational fluoroquinolone with in vitro activity against common skin pathogens and a favorable pharmacokinetic profile. A randomized, double-blind, multicenter study was conducted in 492 patients to compare the safety and efficacy of temafloxacin 600 mg with ciprofloxacin 750 mg for the treatment of bacterial infections of the skin or skin structure. Both drugs were given twice daily for 7-28 days. The most common diagnoses were abscess and superficial skin infection, which were usually caused by staphylococci. In evaluable patients, clinical success (cure plus improvement) rates were 96% in the temafloxacin group and 99% in the ciprofloxacin group. Bacteriologic eradication rates were 95% and 93% in the temafloxacin and ciprofloxacin groups, respectively. Both regimens were well tolerated. Temafloxacin appears to be safe and effective for the management of bacterial infections of the skin and skin structure.

A receptor-specific peptide for imaging infection and inflammation

The chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (fMLP), when radiolabelled, continues to be an attractive agent for imaging infection or inflammation. Previously, several analogues of fMLP have been prepared and radiolabelled using a bifunctional chelating agent conjugation procedure that was relatively long and complex. We have prepared a new analogue of fMLP, TP765, by the addition of 4-aminobutyric acid (4-ABA) and a group of four amino acids, Gly-Gly-d-Ala-Gly, to the carboxy terminus (i.e. to the phenylalanine) of fMLP. The adduct -(4-ABA)-Gly-Gly-d-Ala-Gly- serves as a chelating moiety for strong chelation with 99Tcm. The use of a peptide as a chelating moiety greatly simplified the synthetic procedure and rendered the analogue ready for instant chelation with 99Tcm. HPLC analysis revealed that 99Tcm-TP765 was a single chemical entity that retained biological activity and neutrophil specificity. 99Tcm-TP765 was stable when challenged with strong chelating agents in vitro and had rapid but biphasic blood clearance (αt½ = 7 min, βt½ = 45 min). Approximately 90% of the radioactivity had cleared from circulation within 45 min post-injection and the agent had accumulated in experimental bacterial or sterile abscesses in significantly (P<0.05) higher quantities than the analogues evaluated previously. Generally, the biodistribution pattern of 99Tcm-TP765 was similar to that of other analogues examined and its abscess uptake was independent of the abscess age. In conclusion, a new analogue of fMLP, 99Tcm-TP765, was prepared by a simple procedure. This new analogue has properties similar to those of previously examined analogues used as agents for imaging infection or inflammation.

Development and Validation of a Quantitative, One-Step, Multiplex, Real-Time Reverse Transcriptase PCR Assay for Detection of Dengue and Chikungunya Viruses

ABSTRACT Dengue virus (DENV) and chikungunya virus (CHIKV) are important human pathogens with common transmission vectors and similar clinical presentations. Patient care may be impacted by the misdiagnosis of DENV and CHIKV in areas where both viruses cocirculate. In this study, we have developed and validated a one-step multiplex reverse transcriptase PCR (RT-PCR) to simultaneously detect, quantify, and differentiate between four DENV serotypes (pan-DENV) and chikungunya virus. The assay uses TaqMan technology, employing two forward primers, three reverse primers, and four fluorophore-labeled probes in a single-reaction format. Coextracted and coamplified RNA was used as an internal control (IC), and in vitro-transcribed DENV and CHIKV RNAs were used to generate standard curves for absolute quantification. The diagnostic 95% limits of detection (LOD) within the linear range were 50 and 60 RNA copies/reaction for DENV (serotypes 1 to 4) and CHIKV, respectively. Our assay was able to detect 53 different strains of DENV, representing four serotypes, and six strains of CHIKV. No cross-reactivity was observed with related flaviviruses and alphaviruses, To evaluate diagnostic sensitivity and specificity, 89 clinical samples positive or negative for DENV (serotypes 1 to 4) and CHIKV by the standard virus isolation method were tested in our assay. The multiplex RT-PCR assay showed 95% sensitivity and 100% specificity for DENV and 100% sensitivity and specificity for CHIKV. With an assay turnaround time of less than 2 h, including extraction of RNA, the multiplex quantitative RT-PCR assay provides rapid diagnosis for the differential detection of two clinically indistinguishable diseases, whose geographical occurrence is increasingly overlapping.

Rothia mucilaginosa Prosthetic Device Infections: a Case of Prosthetic Valve Endocarditis

ABSTRACT Rothia mucilaginosa is increasingly recognized as an emerging opportunistic pathogen associated with prosthetic device infections. Infective endocarditis is one of the most common clinical presentations. We report a case of R. mucilaginosa prosthetic valve endocarditis and review the literature of prosthetic device infections caused by this organism.

Fatal Case of Weeksella virosa Sepsis

ABSTRACT Weeksella virosa is an aerobic Gram-negative rod that has rarely been reported to cause infection. We describe a fatal case of W. virosa sepsis in a young female with end-stage renal disease, report three additional cases of W. virosa infection, and review the literature regarding this infection.