D. Jane Hata

Real-Time PCR Method for Detection of Zygomycetes

ABSTRACT Zygomycete infections can be devastating in immunocompromised hosts. Difficulties in the histopathologic differentiation of this class from other filamentous fungi (e.g., Aspergillus spp., Fusarium spp.) may lead to delays in diagnosis and initiation of appropriate treatment, thereby significantly affecting patient outcome. A real-time PCR assay was developed to detect species of the zygomycete genera Absidia, Apophysomyces, Cunninghamella, Mucor, Rhizopus, and Saksenaea in culture and tissue samples. Primers and fluorescence resonance energy transfer hybridization probes were designed to detect a 167-bp conserved region of the multicopy zygomycete cytochrome b gene. A plasmid containing target sequence from Mucor racemosus was constructed as a positive control. The analytical sensitivity of the assay is 10 targets/μl, and a specificity panel consisting of other filamentous fungi, yeasts (Candida spp.), and bacteria demonstrated no cross-reactivity in the assay. The clinical sensitivity and specificity of the assay from culture isolates were 100% (39/39) and 92% (59/64), respectively. Sensitivity and specificity determined using a limited number of fresh tissue specimens were both 100% (2/2). The sensitivity seen with formalin-fixed, paraffin-embedded tissues was 56% (35/62), and the specificity was 100% (19/19). The speed, sensitivity, and specificity of the PCR assay indicate that it is useful for the rapid and accurate detection of zygomycetes.

Rapid identification of bacteria and candida using pna-fish from blood and peritoneal fluid cultures: a retrospective clinical study

BackgroundPeptide nucleic acid fluorescent in situ hybridization (PNA-FISH) is a rapid and established method for identification of Candida sp., Gram positive, and Gram negative bacteria from positive blood cultures. This study reports clinical experience in the evaluation of 103 positive blood cultures and 17 positive peritoneal fluid cultures from 120 patients using PNA-FISH. Our study provides evidence as to potential pharmaceutical cost savings based on rapid pathogen identification, in addition to the novel application of PNA-FISH to peritoneal fluid specimens.MethodsIdentification accuracy and elapsed time to identification of Gram positives, Gram negatives, and Candida sp., isolated from blood and peritoneal fluid cultures were assessed using PNA-FISH (AdvanDx), as compared to standard culture methods. Patient charts were reviewed to extrapolate potential pharmaceutical cost savings due to adjustment of antimicrobial or antifungal therapy, based on identification by PNA-FISH.ResultsIn blood cultures, time to identification by standard culture methods for bacteria and Candida sp., averaged 83.6 hours (95% CI 56.7 to 110.5). Identification by PNA-FISH averaged 11.2 hours (95% CI 4.8 to 17.6). Overall PNA-FISH identification accuracy was 98.8% (83/84, 95% CI 93.5% to 99.9%) as compared to culture. In peritoneal fluid, identification of bacteria by culture averaged 87.4 hours (95% CI −92.4 to 267.1). Identification by PNA-FISH averaged 16.4 hours (95% CI −57.3 to 90.0). Overall PNA-FISH identification accuracy was 100% (13/13, 95% CI 75.3% to 100%). For Candida sp., pharmaceutical cost savings based on PNA-FISH identification could be

Morbidity and mortality among patients with respiratory syncytial virus infection: a 2-year retrospective review

377.74/day. For coagulase-negative staphylococcus (CoNS), discontinuation of vancomycin could result in savings of

Precision across the Analytical Measuring Range of a Quantitative Real-Time PCR Assay for Cytomegalovirus Detection among Three Clinical Laboratories

20.00/day.ConclusionsIn this retrospective study, excellent accuracy of PNA-FISH in blood and peritoneal fluids with reduced time to identification was observed, as compared to conventional culture-based techniques. Species-level identification based on PNA-FISH could contribute to notable cost savings due to adjustments in empiric antimicrobial or antifungal therapy as appropriate to the pathogen identified.

Diagnostic Testing for Zika Virus: a Postoutbreak Update

Previous studies have demonstrated high morbidity and mortality for adult patients with respiratory syncytial virus (RSV) infection. We performed a retrospective, multicenter, two-year chart review of all patients (n = 334) testing positive for RSV by the ProFlu + (®) Influenza A/B and RSV assay (Hologic, Bedford, MA). We analyzed indicators of morbidity and mortality in children <6 years old, immunocompetent and immunosuppressed adults, and transplant patients. Significant morbidity and mortality was observed among hematopoietic stem cell transplant patients (7.3%, 60-day mortality), solid organ transplant patients (13.3%, 60-day mortality), and COPD patients (12.8%, 60-day mortality). Of the patients positive for RSV, 144 (43.1%) of 334 received antibacterials or antifungals following diagnosis. Of these patients, a bacterial or fungal pathogen was not recovered from 60% of cases. Despite advances in RSV treatment, certain populations appear to be inadequately treated, while others appear to be inappropriately treated with unnecessary antimicrobials.

Adult Intestinal Botulism: A Rare Presentation in an Immunocompromised Patient With Short Bowel Syndrome

ABSTRACT This study measured the precision of a quantitative laboratory-developed real-time PCR test for cytomegalovirus performed at three different clinical laboratories that use the same methodology. The overall standard deviation (adjusted for analyte level) was 0.18 log10 copies/ml, and there was no significant relationship between standard deviation and analytical measuring range.

Trichosporon asahii Infection in a Patient with Metastatic Prostate Cancer as an Example of an Emerging Fungal Pathogen

ABSTRACT Since the emergence and dissemination of Zika virus (ZIKV) in late 2015, our understanding of the biology, transmission, clinical disease, and potential sequelae associated with infection has markedly expanded. Over the past 2 years, the number of diagnostic assays for ZIKV has increased from none in 2015 to 5 serological assays and 14 molecular assays in 2017, all with emergency use authorization granted through the U.S. Food and Drug Administration. Here we provide an update on ZIKV, addressing what we have collectively learned since the outbreak began, including a summary of currently available diagnostic assays for this virus.

Molecular Methods for Identification and Characterization of Acinetobacter spp.

The cholinergic heat-labile neurotoxin produced by Clostridium species is primarily responsible for the clinical manifestations of botulism. The classic phenotypic presentation of botulism consists of subacute descending flaccid paralysis with intact sensory function. Traditionally, it is classified into 3 main forms (foodborne, wound-related, and infantile) on the basis of primary site of toxin entry into the human nervous system. Toxemia is the common pathophysiology in all forms of botulism. Adult intestinal toxemia botulism is an extremely rare form of the disease with pathogenesis similar to that of infant-type botulism. Symptomatic adults usually have an anatomic abnormality in the gastrointestinal tract leading to changes in normal gut flora. The current case is an addition to the growing literature on this unusual clinical variant of botulism.

Fastidious and Furious: Reporting Antimicrobial Susceptibility Testing for Fastidious or Infrequently Isolated Bacteria

CLINICAL HISTORY PATIENT 59-year-old white man. CHIEF COMPLAINT Nausea, constant urge to urinate, and intermittent lower back pain that wraps around his right iliac crest and down his right anterior thigh to the level of his right knee. HISTORY OF PRESENT ILLNESS The patient sought radiation oncology consultation for his metastatic prostate cancer. He has had nephrostomy tubes and ureteral stents implanted to help with his bilateral uropathic manifestations. Two days earlier, his ureteral stent was removed and sent for culture during the replacement of his malfunctioning nephrostomy tubes; Trichosporon asahii had been cultured from the stent. PREVIOUS MEDICAL HISTORY Castration-resistant prostate cancer with bone metastasis, left upper abdominal shingles, recurrent urinary tract infections (UTIs), chronic anemia due to chemotherapy, and obstructive bilateral uropathy. FAMILY HISTORY Mother had breast cancer and father had lung cancer and heart disease. PHYSICAL EXAMINATION FINDINGS The patient was alert and oriented. There was a small, soft, compressible nodule, or cyst, in the posterior supraclavicular region. His lungs were clear, and his pulse had a regular rate and rhythm. PRINCIPLE LABORATORY FINDINGS Table 1.

1426Correlation of Cytomegalovirus Viral Load between a Laboratory-Developed Test and a WHO-Calibrated Commercial Assay in Transplant Recipients

This chapter focuses on current challenges in the clinical laboratory setting for identification, susceptibility testing, and epidemiology of Acinetobacter spp. The application of various molecular methods to aid in the generation of accurate information in the healthcare environment is discussed. Acinetobacter is a uniquely successful organism that is now presenting unusual challenges in terms of its detection and treatment in human disease. Classification of species within this genus has historically presented difficulty owing to the genetic diversity of this organism. Application of various molecular typing methods has been instrumental in the definition of the 31 described species of Acinetobacter , as phenotypic methods may not achieve adequate levels of speciation. Based on DNA sequence homology, the A. baumannii–A. calcoaceticus complex consists of four members: A. baumannii , genospecies 3 and 13TU, and A. calcoaceticus . Of these, A. baumannii is most clinically significant in human disease. Acinetobacter spp. has been isolated from extremely diverse environments and is well adapted to multiple sites on the human host. Pneumonia, bacteremia, and wound infections are the most common manifestations of Acinetobacter in the ICU. A. lwoffii is also a significant cause of nosocomial infections. A significant development is the emergence of Acinetobacter strains with resistance to multiple classes of antibiotics.