Buckwalter, Seanne P.

Abstract Introduction/Objective Molecular assays for Bartonella species are important in diagnosing infection and expediting patient treatment. Real time polymerase chain reaction (RT-PCR) using fluorescent resonance energy transfer (FRET) hybridization probes can be used to detect Bartonella species in blood and fresh/fixed tissue biopsies in RT-PCR instruments. Over time, new technologies and reagents are introduced and existing PCR primers and FRET probes must be re-validated on new platforms. This study aimed to compare the performance of a Bartonella RT-PCR assay using the sunsetting Roche LightCycler® 2.0 (Roche Diagnostics, Indianapolis, IN) and newer LightCycler® 480 RT- PCR instruments. Methods/Case Report DNA was extracted from 132 historically positive, whole organism spiked, and historically negative whole blood and formalin fixed paraffin embedded (FFPE) samples. Samples were run on the LightCycler® 2.0 using instrument specific LightCycler® FastStart DNA Master HybProbe enzyme and compared to results generated using the LightCycler® 480 and its instrument specific LightCycler® 480 Genotyping Master enzyme. During optimization, MgCl2 concentrations and thermocycling profiles were adjusted. Accuracy, specificity, inclusivity, and limit of detection studies were performed. Crossing point (Cp), melting temperature (Tm), fluorescent peak and fluorescent background values were compared between the two instruments. Results (if a Case Study enter NA) The agreement in accuracy between the LightCycler® 2.0 and the LightCycler® 480 was 100% for whole blood samples. For historically positive FFPE samples, LightCycler® 2.0 sensitivity and LightCycler® 480 sensitivity were 86% and 100%, respectively. Specificity and inclusivity of the assay were identical between the two instruments. The limit of detection in whole blood was 5-fold lower on the LightCycler® 480 (50 copies/µL) compared to the LightCycler® 2.0 (250 copies/µL). Mean Cp and fluorescent peak intensity values increased by 5.1% and 65-fold, respectively. Conclusion The study demonstrates similar performance and improved limit of detection for the Bartonella FRET hybridization probe RT-PCR assay on the LightCycler® 480 compared to the LightCycler® 2.0.

Rapid and accurate diagnostic testing is essential to bring the ongoing COVID-19 pandemic to an end. As the demand for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) testing continues to increase amid supply shortages, many laboratories have investigated the use of sources other than nasopharyngeal (NP) swabs.

Candida auris is an emerging fungal pathogen with cases reported in countries around the world and in 19 states within the United States as of August 2020. The CDC has recommended that hospitals perform active surveillance upon admission for patients with the appropriate risk factors. Currently, active surveillance requires that local hospitals send surveillance swabs to a public health laboratory for analysis. In this work, a real-time PCR assay was developed for the specific detection of C. auris from surveillance swabs, blood, and urine to enable rapid detection of this pathogen. The assay uses commercially available primers and reporter probes and it was verified on the LightCycler 480 PCR platform. Contrived specimens and prospectively collected composite groin/axilla surveillance swabs were used to validate the assay. The performance of the PCR assay on surveillance swabs was also compared to a second PCR assay targeting C. auris that was performed at the Minnesota Department of Health–Public Health Laboratory (MDH-PHL). Our PCR assay is able to detect and differentiate C. auris from closely related Candida species such as C. duobushaemulonii, C. haemulonii, and C. pseudohaemulonii on the basis of melting curve temperature differences.

AbstractBackgroundGlobicatella sanguinis is an uncommon pathogen that may be misdiagnosed as viridans group streptococci. We review the literature of Globicatella and report 2 clinical cases in which catalase-negative Gram-positive cocci resembling viridans group streptococci with elevated minimum inhibitory concentrations (MICs) to ceftriaxone were inconsistently identified phenotypically, with further molecular characterization and ultimate identification of G sanguinis.MethodsTwo clinical strains (from 2 obese women; 1 with a prosthetic hip infection and the other with bacteremia) were analyzed with standard identification methods, followed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, 16S recombinant ribonucleic acid (rRNA), and sodA polymerase chain reaction (PCR). The existing medical literature on Globicatella also was reviewed.ResultsStandard phenotypic methods failed to consistently identify the isolates. 16S PCR yielded sequences that confirmed Globicatella species. sodA sequencing provided species-level identification of G sanguinis. The review of literature reveals G sanguinis as an increasingly reported cause of infections of the urine, meninges, and blood. To our knowledge, this is the first reported case of an orthopedic infection caused by Globicatella sanguinis. A review of the 37 known cases of G sanguinis infection revealed that 83% of patients are female, and 89% are at the extremes of age (<5 or >65 years).ConclusionsGlobicatella sanguinis, an uncommon pathogen with elevated minimum inhibitory concentrations to third-generation cephalosporins, is difficult to identify by phenotypic methods and typically causes infections in females at the extremes of age. It may colonize skin or mucosal surfaces. Advanced molecular techniques utilizing 16S rRNA with sodA PCR accurately identify G sanguinis.

ABSTRACT The value of matrix-assisted laser desorption ionization−time of flight mass spectrometry (MALDI-TOF MS) for the identification of bacteria and yeasts is well documented in the literature. Its utility for the identification of mycobacteria and Nocardia spp. has also been reported in a limited scope. In this work, we report the specificity of MALDI-TOF MS for the identification of 162 Mycobacterium species and subspecies, 53 Nocardia species, and 13 genera (totaling 43 species) of other aerobic actinomycetes using both the MALDI-TOF MS manufacturer's supplied database(s) and a custom database generated in our laboratory. The performance of a simplified processing and extraction procedure was also evaluated, and, similar to the results in an earlier literature report, our viability studies confirmed the ability of this process to inactivate Mycobacterium tuberculosis prior to analysis. Following library construction and the specificity study, the performance of MALDI-TOF MS was directly compared with that of 16S rRNA gene sequencing for the evaluation of 297 mycobacteria isolates, 148 Nocardia species isolates, and 61 other aerobic actinomycetes isolates under routine clinical laboratory working conditions over a 6-month period. MALDI-TOF MS is a valuable tool for the identification of these groups of organisms. Limitations in the databases and in the ability of MALDI-TOF MS to rapidly identify slowly growing mycobacteria are discussed.

ABSTRACT We present a case of tenosynovitis caused by a novel, slowly growing, nonchromogenic, nontuberculous mycobacterium (NTM). Originally misidentified as Mycobacterium tuberculosis complex, the NTM cross-reacts with the M. tuberculosis complex nucleic acid hybridization probe, a M. tuberculosis gamma interferon release assay, and is closely related to M. tuberculosis by 16S rRNA gene sequencing.