Temporal trends in antifungal susceptibility of Cryptococcus neoformans isolates from a reference laboratory in the United States, 2011-2021.

BACKGROUND: There are no established clinical breakpoints for antifungal agents against Cryptococcus species; however, epidemiological cut-off values can help distinguish wild-type (WT) isolates without any acquired resistance from non-WT strains, which may harbour resistance mechanisms. PATIENTS/METHODS: We describe the trends of antifungal MICs and percentages of WT C. neoformans species complex (CNSC) isolates processed in our reference laboratory from November 2011 to June 2021. There were only nine isolates in 2011, thus, we included them in the year 2012 for data analysis. Clinical data is also described when available. RESULTS: We identified 632 CNSC, the majority collected from blood (n = 301), cerebrospinal fluid (n = 230), and respiratory (n = 71) sources. The overall percentage of WT isolates for amphotericin B (AMB), 5-flucytosine, and fluconazole was 77%, 98%, and 91%, respectively. We noticed a statistically significant change in the percentage of AMB WT isolates over the years, with 98% of isolates being WT in 2012 compared to 79% in 2021 (p < .01). A similar change was not observed for other antifungal agents. Clinical data was available for 36 patients, primarily non-HIV immunocompromised patients with disseminated cryptococcosis. There were no statistically significant differences in the clinical characteristics and outcomes between patients with WT (58.3%) versus non-WT (41.7%) isolates, but we noticed higher mortality in patients infected with an AMB non-WT CNSC isolate. CONCLUSIONS: We observed an increase in the percentage of AMB non-WT CNSC isolates in the past decade. The clinical implications of this finding warrant further evaluation in larger studies.

Description, validation, and review of a decade of experience with a laboratory-developed PCR test for detection of Mycobacterium tuberculosis complex in pulmonary and extrapulmonary specimens.

Rapid detection of Mycobacterium tuberculosis complex directly from clinical specimens is critical for patient care. Mycobacterial culture requires days to weeks for results and therefore many laboratories employ rapid molecular methods for the diagnosis of tuberculosis. There are two FDA-cleared molecular assays for the detection of M. tuberculosis complex in the United States and both are cleared for testing of respiratory specimens only. The detection of M. tuberculosis complex in extrapulmonary specimens is often done using laboratory-developed PCR methods. In this work, the verification and subsequent validation of test performance over a decade is detailed for a laboratory-developed PCR assay (MTBRP) that detects M. tuberculosis complex from respiratory and non-respiratory specimens. The assay also provides information about potential isoniazid resistance. The performance of the MTBRP PCR assay was compared to the Cepheid Xpert MTB/RIF assay in acid-fast smear positive and smear negative specimens and mycobacterial culture for acid-fast smear positive specimens. The MTBRP assay demonstrated 99% correlation with the Xpert MTB/RIF assay using 499 respiratory specimens. The performance of the MTBRP PCR assay compared with mycobacterial culture for 867 AFB smear positive respiratory and non-respiratory specimens demonstrated a sensitivity of 100% and a specificity of 99.1%. This work provides longitudinal evidence using real-world clinical laboratory conditions and specimens to demonstrate that laboratory-developed PCR assays such as the MTBRP can provide a rapid and sensitive method for detection of pulmonary and extra-pulmonary tuberculosis from a wide-variety of smear positive specimen sources.

Mycobacterium septicum: a 6-Year Clinical Experience from a Tertiary Hospital and Reference Laboratory.

Mycobacterium septicum is a rarely identified nontuberculous mycobacterium capable of causing infections in both healthy and immunocompromised individuals. Only a few cases of M. septicum infections have been reported, which makes recognizing corresponding clinical disease more challenging for clinicians. Antimicrobial susceptibility profiles for this organism are not well described, and corresponding optimal therapeutic regimens have not been established. We report a tertiary care center's experience with M. septicum from 2014 to 2020. Twelve adult patients with positive cultures for M. septicum were identified. Most cases were identified from sputum samples of individuals with underlying lung disease. Most cases involving M. septicum isolation in culture were not felt to be clinically significant. Two cases were considered possible infections, while only one case was considered a definite infection that required antimicrobial treatment. All M. septicum isolates were susceptible in vitro to amikacin, ciprofloxacin, imipenem, linezolid, moxifloxacin, and trimethoprim-sulfamethoxazole. Isolates were universally resistant to clarithromycin and doxycycline. The isolation of M. septicum in culture is uncommon and requires clinical correlation to determine its clinical relevance and need for treatment. Susceptibility testing should be performed to guide therapy.

Utility of Mycobacterium tuberculosis PCR in ruling out active disease and impact on isolation requirements in a low prevalence setting.

OBJECTIVE: To analyze and interpret clinical microbiology data for specimens tested with the fluorochrome stain (AFB stain), mycobacterial culture and a laboratory-developed Mycobacterium tuberculosis (MTB) PCR in order to understand the performance of each test and to demonstrate the utility of MTB PCR to assist with decisions regarding discontinuation of airborne isolation. METHODS: Retrospective cohort analysis of 2798 respiratory specimens from 2006 patients in the period between November 1st, 2011 and January 1st, 2018. RESULTS: 53.7% were males, median age was 61 years, and 43 patients were HIV positive. Results demonstrated positive mycobacterial cultures for MTB in 52 specimens (1.9%) and for nontuberculous mycobacteria (NTM) or aerobic actinomycetes (eg., Nocardia spp.) in 435 specimens (16%). Using mycobacterial culture as the gold standard, AFB smear had a sensitivity of 48.1% while MTB PCR had a sensitivity of 96.0% in AFB smear positive specimens and an overall sensitivity of 57.7% with PPV of 94% and a NPV of 99%. CONCLUSIONS: The combination of a positive AFB smear with a negative MTB PCR offers a rapid result to rule out active pulmonary MTB in a low prevalence setting. In this study, that combination reliably excluded active tuberculosis (NPV of 99.2%). The combination of a positive AFB smear with a negative MTB PCR indicated pulmonary NTM infection with the results available within 1 day. There was little benefit to pursuing collection and testing of more than 2 respiratory specimens in a low prevalence setting for both long term diagnostic or rapid isolation discontinuation purposes.

Retrospective Analysis of Antimicrobial Susceptibility Profiles of Nocardia Species from a Tertiary Hospital and Reference Laboratory, 2011 to 2017.

Nocardia species are found worldwide and are opportunistic pathogens of both immunocompromised and immunocompetent hosts. Recent updates to the taxonomy of this genus have indicated that there are more than 90 recognized species of Nocardia with 54 species reported to be clinically relevant. In this paper, we report the species distribution, specimen source distribution, and antimicrobial susceptibility profiles of 2,091 clinical isolates recovered for the years 2011 to 2017 using the updated taxonomy. The most commonly isolated species included Nocardia nova complex, Nocardia cyriacigeorgica, and Nocardia farcinica complex, with an additional 25 species or species complexes recovered from clinical specimens. The antimicrobial susceptibility profile was highly variable between the species, but in general, amikacin, linezolid, and trimethoprim-sulfamethoxazole demonstrated good in vitro activity against most species.

Automated broth-based systems versus the MYCOTB plate for antimicrobial susceptibility testing of the Mycobacterium tuberculosis complex: challenges in interpretation.

We examined categorical agreement between automated mycobacterial susceptibility testing methods (Mycobacterial Growth Indicator Tube [MGIT] 960 System and the VersaTREK Mycobacteria Detection and Susceptibility System) which are based on single critical concentration (CC) "breakpoints" and a commercial microbroth dilution method (Sensititre Mycobacterium tuberculosis MIC Plate [MYCOTB]) which provides an MIC value. Mycobacterium tuberculosis isolates (n=355) were tested against three first-line antimycobacterial agents (ethambutol [EMB], isoniazid [INH], rifampin [RIF]) using the MYCOTB plate and either the MGIT 960 (site 1, n=142) or VersaTREK (site 2, n=213) systems. Overall categorical agreement was 96.8%. When stratified by drug and CC-defined susceptible and resistant isolates, concordance ranged from 75% to 100%. Interpretation of MIC-based results versus established CC-based results was challenging for drugs whose CC was not represented by an exactly equivalent concentration in the manufacturer-defined dilutions on the MYCOTB plate (EMB, INH). We propose interpretations of MYCOTB plate MICs using the currently available plate configuration.

Mycobacterium arupense flexor tenosynovitis: case report and review of antimicrobial susceptibility profiles for 40 clinical isolates.

We describe a case of chronic tenosynovitis in the hand of a 58-year-old cattle farmer. Surgical biopsy specimens grew Mycobacterium arupense. The patient responded to surgery and antimicrobial therapy based on in vitro susceptibility testing. The antimicrobial susceptibility profiles of the isolate from this patient and 39 additional clinical isolates are presented.

Evaluation of the Yeast Traffic Light PNA FISH probes for identification of Candida species from positive blood cultures.

The Yeast Traffic Light PNA FISH kit (YTL) correctly identified Candida spp. in 207/216 (96%) positive blood cultures. Discordant results were seen with known cross-reacting species and cultures containing Candida lambica and Rhodotorula mucilaginosa. The YTL provides rapid, reliable identification of the five common Candida species found in blood cultures.

Evaluation of Mycobacterium avium complex clarithromycin susceptibility testing using SLOMYCO Sensititre panels and JustOne strips.

The SLOMYCO Sensititre panel and the custom JustOne strip (both from TREK Diagnostic Systems, Cleveland, OH) were evaluated for susceptibility testing of Mycobacterium avium complex isolates against clarithromycin. Seventy-one archived and prospectively collected isolates were tested using both the SLOMYCO panel and the JustOne strip, and the results were compared to those obtained using the BACTEC 460 (BD, Sparks, MD) radiometric method and a broth microdilution reference method. Results obtained by the SLOMYCO panel and the JustOne strip agreed with the BACTEC 460 method for 64/71 isolates (90%). Similarly, concordance with the broth microdilution method was 40/43 isolates (93%) for both test systems. The effect of the source medium on inoculum preparation was evaluated, and there were no differences noted in MICs, regardless of whether the inoculum was prepared from isolates grown in Middlebrook 7H9 medium, on Middlebrook 7H10 agar, or in VersaTREK broth culture bottles (Trek Diagnostics). Clarithromycin susceptibility testing of MAC using the SLOMYCO panel and the JustOne strip methods is easy to set up and simple to read and is readily incorporate into the clinical laboratory. These systems offer advantages over the BACTEC 460 system including the lack of a need for radioactive substrates, sharps, or costly instrumentation.