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

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.

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.

Infections in Heart and Lung Transplant Recipients.

Patients undergoing thoracic organ transplantation procedures involving the heart or lung are at increased risk for developing a wide variety of infections due to their underlying immunosuppression and/or other factors. Lung transplant recipients are at high risk for developing infections caused by bacteria, viruses, and opportunistic fungi, whereas heart transplant recipients are at risk for developing infections caused by these same microorganisms, as well as parasitic infections, including toxoplasmosis and New World trypanosomiasis. This review will highlight the various infections that thoracic organ transplant recipients may develop following their procedures.

Sterility of the personal protection system in total joint arthroplasty.

BACKGROUND: Bacteria shed by operating room personnel is a source of wound contamination and postoperative infections. The personal protection system (PPS) was designed to decrease airborne bacteria and intraoperative contamination in total joint arthroplasty. QUESTIONS/PURPOSES: We determined the microbial contamination rate of the PPS and incidence of contamination with key pathogens, Staphylococcus aureus and coagulase-negative staphylococci. PATIENTS AND METHODS: We prospectively evaluated PPS contamination in 61 primary THAs and 41 TKAs. The PPS were assumed to be sterile before opening the packs. The initial culture was taken immediately after the hood was placed over the helmet. Four cultures were collected at the conclusion of the procedure. Plates were examined and colonies were classified according to Gram stain results and biochemical tests. S. aureus was classified as methicillin-resistant or -susceptible. RESULTS: At time zero, 22 of 102 cultures isolated an organism, accounting for a contamination rate of 22%. The bacterial contamination rate of the PPS at the conclusion of the procedure was 47% (48 of 102). The relative percentage of the various organisms found was coagulase-negative staphylococci 50%, Micrococcus sp. 20%, methicillin-susceptible S. aureus 11%, and methicillin-resistant S. aureus (MRSA) 1%. CONCLUSIONS: The external surface of the PPS cannot be assumed to be sterile after its removal from the original packaging. Of all the PPS studied, the potential pathogens coagulase-negative staphylococcus, S. aureus, and MRSA were found in 43%. This study supports the need to change gloves if the PPS is touched or adjusted during the procedure.

An outbreak of Fusarium keratitis associated with contact lens use in the northeastern United States.

PURPOSE: To report an outbreak of Fusarium keratitis in contact lens (CL) wearers in the northeastern United States. METHODS: Over a 41-month period, all cases with culture-proven corneal ulceration secondary to Fusarium at 2 tertiary care eye centers were identified through the microbiology departments of each institution, and a retrospective review of charts was performed. Statistical analyses were performed to evaluate a possible association of Fusarium keratitis with specific CL and CL solution brands. RESULTS: Fifteen cases of Fusarium keratitis were reported at the 2 tertiary centers between July 2005 and May 2006 (16.4 cases/yr) compared with 6 cases over the previous 30 months from January 2003 to June 2005 (2.4 cases/yr). All 15 of the more recent cases were CL users, and none had a history of trauma. All 15 patients claimed use of ReNu brand contact lens solution when they developed keratitis. Twelve (80.0%) of 15 patients were Acuvue soft contact lens users. Ten (66.7%) of 15 patients used tap water to rinse their contact lens cases. Six (40.0%) of 15 cases have thus far required corneal transplantation. CONCLUSIONS: The incidence of corneal ulceration secondary to Fusarium has increased sevenfold over the reported 11-month period at 2 tertiary eye care centers in the northeastern United States compared with the previous 30 months. There seems to be an association between the recent outbreak of Fusarium keratitis among CL users and the use of ReNu contact lens solution. Medical treatment of Fusarium keratitis may be ineffective, and emergent penetrating keratoplasty (PKP) may be required in some patients. CL users and their physicians should reconsider the risks of CL use and discuss proper lens care techniques.

Chikungunya Virus.

For chikungunya virus (CHIKV), the long-term sequelae from infection are yet ill-defined. The prolonged debilitating arthralgia associated with CHIKV infection has tremendous potential for impacting the global economy and should be considered when evaluating the human burden of disease and the allocation of resources. There is much still unknown about CHIKV and the illnesses that it causes. Developing a better understanding of the pathogenesis of CHIKV infection is a priority and forms the basis for developing effective strategies at infection prevention and disease control.

Clinical and Serological Insights from the Asian Lineage Chikungunya Outbreak in Grenada, 2014: An Observational Study.

Chikungunya virus (CHIKV) spread rapidly throughout the Caribbean region in 2014, and the first serologically confirmed case was seen in Grenada in July. This study investigated the outbreak of CHIKV in Grenada to identify the distinguishing clinical manifestations and the symptoms that corresponded the closest with serological test results. Sera were tested by IgM enzyme-linked immunosorbent assay and polymerase chain reaction to distinguish between cases positive or negative for CHIKV. Of 493 cases, 426 (86%) tested positive for CHIKV. The diagnostic decision rule, "Define as CHIKV positive a patient presenting with joint pain and any combination of fever, body pain, or rash," produced the closest agreement (85%) with the serological test results (Cohen's kappa, k = 0.289, P value < 0.001). When laboratory facilities are not available for diagnostic confirmation, syndromic surveillance using these four symptoms could be useful to define cases during a CHIKV outbreak when CHIKV is the predominant circulating arbovirus.

Prevalence and acquisition of vancomycin-resistant enterococci in a medical intensive care unit.

Patients admitted to a medical intensive care unit during 21 days had rectal and urine samples cultured for vancomycin-resistant enterococci (VRE). The prevalence rate was 55.3%. Only enteral feedings were associated with acquisition of VRE. One-quarter of VRE transmission resulted from acquisition in the study unit, 35% arrived from other units, and 15% from other hospitals.

Apoptotic abscess imaging with 99mTc-HYNIC-rh-Annexin-V.

Abscess formation causes systemic and localized up-regulation of neutrophil [polymorphonuclear leukocytes (PMNs)] signaling pathways. In the abscess, following bacterial ingestion or PMN activation by inflammatory mediators, PMN apoptosis is elevated and leads to the externalization of phosphatidylserine. Annexin-V (AnxV) has been shown to have high affinity to externalized phosphatidylserine. We hypothesized that (99m)Tc-AnxV will target high densities of apoptotic PMNs and image abscesses. AnxV, conjugated with hydrazinenicaotinamide (HYNIC), was labeled with reduced (99m)TcO(4)(-) and its purity was determined by instant thin-layer chromatography. Apoptosis was induced in isolated human PMNs by incubation in 2% saline for 17 and 22 h at 37 degrees C. PMNs were then incubated with (99m)Tc-HYNIC-AnxV and associated (99m)Tc was determined. Abscesses were induced in mice by intramuscular injection of bacteria or turpentine. Following intravenous administration of (99m)Tc-HYNIC-AnxV, mice were imaged and tissue distribution studied at 4 and 24 h. Radiochemical purity of (99m)Tc-HYNIC-AnxV was 84.9+/-8.11%. At 17 h, (99m)Tc-HYNIC-AnxV bound to apoptotic PMNs was 71.6+/-0.01% and 48.6+/-0.01% for experimental and control cells, respectively (P=.002). At 22 h, experimental cells retained 74.9+/-0.02% and control cells retained 47.2+/-0.02% (P=.005). (99m)Tc-HYNIC-AnxV associated with bacterial abscesses was 1.25+/-0.09 and 3.75+/-0.83 percent injected dose per gram (%ID/g) at 4 and 24 h compared to turpentine abscesses which was 1.02+/-0.16 and 0.72+/-0.17 %ID/g at 4 (P

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.

Post-traumatic fungal keratitis caused by Carpoligna sp.

PURPOSE: To report the first case of fungal keratitis caused by presumed Carpoligna species. METHODS: A 37-year-old gardener sustained a full-thickness, stellate corneal laceration while cutting wood outdoors with a circular saw. Two months after surgical repair, he developed a severe infectious keratitis with descemetocoele at the apex of the original stellate laceration. RESULTS: Culture results confirmed fungal elements without evidence of bacteria. Oral and topical voriconazole were initiated. Due to compliance and cost issues, voriconazole was replaced with natamycin 5% prior to discharge from hospital. The patient improved and healed without perforation. The patient was left with a central stromal scar. DNA extraction from the fungal colony allowed PCR amplification of the 28s ribosomal RNA region of the fungus that led to the diagnosis of Carpoligna pleurothecii. Corticosteroids were never used during the patient's treatment. CONCLUSION: This is the first reported case of infectious keratitis caused by presumed Carpoligna species. The treatment for Carpoligna pleurothecii keratitis includes voriconazole, natamycin, and possibly amphotericin B.

Curvularia fungi presenting as a large cranial base meningioma: case report.

OBJECTIVE: Fungal infections are emerging as a growing threat to human health, especially in immunocompromised patients. Candida, Cryptococcus, and Aspergillus are a few of the commonly encountered organisms leading to brain abscesses. In this report, we describe Curvularia geniculata as the causative agent in central nervous system infection. CLINICAL PRESENTATION: Our review of the literature did not reveal a similar published case of central nervous system infection with this organism. A 35-year-old African-American man presented with obstructive hydrocephalus from a large cranial base lesion. Imaging characteristics on computed tomographic and magnetic resonance imaging scans were consistent with those of a cranial base meningioma. INTERVENTION: The patient underwent an endoscopic transnasal/transclival approach to the anterior middle cranial base for biopsy and decompression of this lesion. A spindle cell proliferation was observed on frozen section, which favored a diagnosis of meningioma. However, on permanent sections, we identified fungal hyphae with budding. Subsequent biopsies grew Curvularia in fungal cultures. Deoxyribonucleic acid sequencing was used to confirm the identification of the isolate as Curvularia geniculata. CONCLUSION: Limited data are available for in vitro susceptibility testing of Curvularia, and treatment modalities have not yet been standardized. The prognosis is usually poor. Despite being treated with voriconazole and intravenous amphotericin, this patient progressed to multiorgan failure and ultimately died. This is the first reported case of central nervous system infection by Curvularia geniculata.

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.