Progressive disseminated histoplasmosis: The experience in one non-endemic medical center.

Histoplasmosis, the most common endemic mycosis in North America, presents in a myriad of ways, spanning the spectrum from self-limiting pneumonia to progressive disseminated histoplasmosis (PDH). Toward better describing contemporary histoplasmosis syndromes, risks, and outcomes, this single-center retrospective cohort study was performed (2009-2019). The population who developed PDH was similar to that with other forms of histoplasmosis (OFH) except for higher rates of preexisting immunocompromising conditions (91.3% vs. 40%, P < .001) and a trend toward receiving more chronic immunosuppression (65.2% vs. 33.3%, P = .054) compared to those with OFH. Diagnosis was most frequently achieved by urinary or serum antigen positivity. People with PDH more frequently tested positive compared to those with OFH, but negative tests did not rule out histoplasmosis. Median time to diagnosis was prolonged among people with both PDH and OFH (32 vs. 31 days, respectively). Following diagnosis, people with PDH received more liposomal amphotericin (78.3% vs. 20%, P < .001). Subsequent survival at 90 and 365 days and treatment response were similar in both groups. Patients with PDH were more often hospitalized (95.7% vs. 60%, P = .006); however, once admitted, there were no differences in hospital length of stay or intensive care unit admission rate. The challenges of diagnosing histoplasmosis based on clinical presentation alone highlight the need for heightened awareness of these entities especially given the recent reports on expanded endemicity and delays in diagnosis.

Histoplasmosis is the most common endemic mycosis in North America. This article summarizes the clinical features, risk factors, and outcomes in patients who developed disseminated disease compared to more localized forms of histoplasmosis.

Clinical Validation of the Xpert MTB/RIF Test for Identification of the Mycobacterium tuberculosis Complex in Acid-Fast Bacillus Smear-Positive MGIT Broth Cultures.

The identification of the Mycobacterium tuberculosis complex (MTBC) from smear-positive broth cultures can be achieved using several methods, including both lab-developed and commercially available molecular assays. In the United States, a commercially available probe-based assay has been used for over a decade by many laboratories for identification of MTBC directly from acid-fast bacilli (AFB) smear-positive broth cultures, including those recovered from the MGIT 960 system. However, recent difficulties in obtaining probe kits for identification resulted in mycobacteriology laboratories looking for alternative platforms to provide for rapid identification of MTBC and detection of rifampin resistance. The Xpert MTB/RIF test (Cepheid, Sunnyvale, CA) has shown high sensitivity for the diagnosis of MTBC from pulmonary specimens but is not often used for identification directly from smear-positive MGIT 960 broth cultures (Becton, Dickinson, Sparks, MD). We sought to validate the Xpert MTB/RIF test for use with AFB smear-positive MGIT 960 cultures in a clinical hospital setting. Overall, the assay showed a categorical agreement of 100% for identification of MTBC and detection of rifampin resistance. No false-positive results or cross-reactivity were noted. Findings indicate that the Xpert MTB/RIF test may be suitable as a rapid replacement for identification of MTBC and detection of rifampin resistance from AFB smear-positive MGIT 960 broth cultures.

A Case of Scedosporium aurantiacum Infection in the United States.

Scedosporium aurantiacum is one of the emergent opportunistic fungal pathogens among immunocompromised hosts. Colonization of S. aurantiacum can also occur in patients with underlying lung diseases such as cystic fibrosis. S. aurantiacum is highly resistant to multiple antifungal agents, and management of the infected patients can be very challenging compared to those infected with other species of Scedosporium. Clinical cases have been geographically restricted mostly in Australia with a small number of cases identified in Europe and Japan. Although clinical isolates of S. aurantiacum from the USA have been included in several research studies, no clinical case of S. aurantiacum infection from the USA has been described. We report a case of S. aurantiacum infection acquired in the SA. Awareness of S. aurantiacum among healthcare providers and the species-level identification for Scedosporium are critically important for appropriate selection of antifungal agents and improvement of treatment outcomes.

Antifungal Susceptibility Profiles and Drug Resistance Mechanisms of Clinical Lomentospora prolificans Isolates.

Lomentospora prolificans is an opportunistic fungal pathogen with low susceptibility to current antifungal drugs. Here, we tested the in vitro susceptibility of 8 drugs against 42 clinical L. prolificans isolates. All isolates showed high MICs to voriconazole (MIC90>16 µg/ml), itraconazole (MIC90>16 µg/ml), posaconazole (MIC90>16 µg/ml), isavuconazole (MIC90>16 µg/ml), amphotericin B (MIC90>16 µg/ml), and terbinafine (MIC90>64 µg/ml) and high minimum effective concentrations (MECs) to micafungin (MEC90>8 µg/ml), with the exception of miltefosine showing an MIC90 value of 4 µg/ml. We examined six different in vitro drug combinations and found that the combination of voriconazole and terbinafine achieved the most synergistic effort against L. prolificans We then annotated the L. prolificans whole genome and located its Cyp51 and Fks1 genes. We completely sequenced the two genes to determine if any mutation would be related to azole and echinocandin resistance in L. prolificans We found no amino acid changes in Cyp51 protein and no tandem repeats in the 5' upstream region of the Cyp51 gene. However, we identified three intrinsic amino acid residues (G138S, M220I, and T289A) in the Cyp51 protein that were linked to azole resistance. Likewise, two intrinsic amino acid residues (F639Y, W695F) that have reported to confer echinocandin resistance were found in Fks1 hot spot regions. In addition, three new amino acid alterations (D440A, S634R, and H1245R) were found outside Fks1 hot spot regions, and their contributions to echinocandin resistance need future investigation. Overall, our findings support the notion that L. prolificans is intrinsically resistant to azoles and echinocandins.

Multicenter Evaluation of a PCR-Based Digital Microfluidics and Electrochemical Detection System for the Rapid Identification of 15 Fungal Pathogens Directly from Positive Blood Cultures.

Routine identification of fungal pathogens from positive blood cultures by culture-based methods can be time-consuming, delaying treatment with appropriate antifungal agents. The GenMark Dx ePlex investigational use only blood culture identification fungal pathogen panel (BCID-FP) rapidly detects 15 fungal targets simultaneously in blood culture samples positive for fungi by Gram staining. We aimed to determine the performance of the BCID-FP in a multicenter clinical study. Blood culture samples collected at 10 United States sites and tested with BCID-FP at 4 sites were compared to the standard-of-care microbiological and biochemical techniques, fluorescence in situ hybridization using peptide nucleic acid probes (PNA-FISH) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Discrepant results were analyzed by bi-directional PCR/sequencing of residual blood culture samples. A total of 866 clinical samples, 120 retrospectively and 21 prospectively collected, along with 725 contrived samples were evaluated. Sensitivity and specificity of detection of Candida species (C. albicans, C. auris, C. dubliniensis, C. famata, C. glabrata, C. guilliermondii, C. kefyr, C. krusei, C. lusitaniae, C. parapsilosis, and C. tropicalis) ranged from 97.1 to 100% and 99.8 to 100%, respectively. For the other organism targets, sensitivity and specificity were as follows: 100% each for Cryptococcus neoformans and C. gattii, 98.6% and 100% for Fusarium spp., and 96.2% and 99.9% for Rhodotorula spp., respectively. In 4 of the 141 clinical samples, the BCID-FP panel correctly identified an additional Candida species, undetected by standard-of-care methods. The BCID-FP panel offers a faster turnaround time for identification of fungal pathogens in positive blood cultures that may allow for earlier antifungal interventions and includes C. auris, a highly multidrug-resistant fungus.

An analytic feasibility study of the BD MAX™ MDR-TB assay for testing of non-sputum specimens for detection of the Mycobacterium tuberculosis complex (MTBC) and isoniazid (INH) and rifampin (RIF) resistance.

Rapid diagnosis of tuberculosis and drug resistance in extrapulmonary specimens can be challenging. The BD MAX™ multidrug resistant (MDR)-TB assay (BD MAX™) has demonstrated high sensitivity and specificity for the detection of the Mycobacterium tuberculosis complex (MTBC) as well as resistance to INH and Rifampin (RIF) in pulmonary specimens but has not been rigorously assessed in extrapulmonary samples. We evaluated the diagnostic accuracy of the BD MAX™ assay for the detection of MTBC and drug resistance in extrapulmonary specimens spiked with MTBC from the Johns Hopkins strain collection. A total of 1083 tests were performed across multiple sample types, with an overall percent agreement of 94.8% (795/839) for detection of MTBC and 99% (379/383) and 96.4% (323/335) for determination of INH and RIF resistance-conferring mutations, respectively. The BD MAX™ assay provides same day detection of MTBC and drug-resistance results and could be a beneficial diagnostic test in extrapulmonary sample types.

Dual Disseminated Aspergillosis and Mucormycosis Diagnosed at Autopsy: A Report of Two Cases of Coinfection and a Review of the Literature.

Coinfection with invasive aspergillosis and mucormycosis in immunocompromised patients has been reported but is rarely confirmed by tissue histology or autopsy. Serum fungal biomarkers and culture are the primary diagnostic tools but are suboptimal for detecting fungal coinfection. Here, we present the cases of two patients who were immunocompromised due to hematologic malignancy where disseminated aspergillosis and mucormycosis coinfection was only diagnosed upon autopsy despite extensive fungal diagnostic workup, and also review recent literature of such instances of coinfection.

Streamlined instrument-free lysis for the detection of Candida auris.

The continued spread of Candida auris in healthcare facilities has increased the demand for widely available screening to aid in containment and inform treatment options. Current methods of detection can be unreliable and require bulky and expensive instruments to lyse and identify fungal pathogens. Here, we present a quick, low-cost, instrument-free method for lysis of C. auris suitable for streamlined sample processing with polymerase chain reaction (PCR) detection. Chemical, thermal, and bead beating lysis techniques were evaluated for lysis performance and compatibility with nucleic acid extraction and downstream PCR reactions. Using only 10 s of manual shaking with glass beads, this method demonstrated a limit of detection (LOD) of C. auris at 500 colony forming units per mL, a 20-fold improvement compared to the LOD without manual shaking, and a 60-fold reduction in time compared to common fungal lysis kits, all while maintaining repeatability and reproducibility across multiple users. This work highlights a simple method for increasing sensitivity and reducing turnaround time of PCR-based C. auris detection and exhibits promise for integration into point-of-care platforms towards real-time triage of colonized patients.

Biochemical Analysis of CaurSOD4, a Potential Therapeutic Target for the Emerging Fungal Pathogen Candida auris.

Candida auris is an emerging multidrug-resistant fungal pathogen. With high mortality rates, there is an urgent need for new antifungals to combat C. auris. Possible antifungal targets include Cu-only superoxide dismutases (SODs), extracellular SODs that are unique to fungi and effectively combat the superoxide burst of host immunity. Cu-only SODs are essential for the virulence of diverse fungal pathogens; however, little is understood about these enzymes in C. auris. We show here that C. auris secretes an enzymatically active Cu-only SOD (CaurSOD4) when cells are starved for Fe, a condition mimicking host environments. Although predicted to attach to cell walls, CaurSOD4 is detected as a soluble extracellular enzyme and can act at a distance to remove superoxide. CaurSOD4 selectively binds Cu and not Zn, and Cu binding is labile compared to bimetallic Cu/Zn SODs. Moreover, CaurSOD4 is susceptible to inhibition by various metal-binding drugs that are without effect on mammalian Cu/Zn SODs. Our studies highlight CaurSOD4 as a potential antifungal target worthy of consideration.

A Portable Droplet Magnetofluidic Device for Point-of-Care Detection of Multidrug-Resistant Candida auris.

Candida auris is an emerging multidrug-resistant fungal pathogen that can cause severe and deadly infections. To date, C. auris has spurred outbreaks in healthcare settings in thirty-three countries across five continents. To control and potentially prevent its spread, there is an urgent need for point-of-care (POC) diagnostics that can rapidly screen patients, close patient contacts, and surveil environmental sources. Droplet magnetofluidics (DM), which leverages nucleic acid-binding magnetic beads for realizing POC-amenable nucleic acid detection platforms, offers a promising solution. Herein, we report the first DM device-coined POC.auris-for POC detection of C. auris. As part of POC.auris, we have incorporated a handheld cell lysis module that lyses C. auris cells with 2 min hands-on time. Subsequently, within the palm-sized and automated DM device, C. auris and control DNA are magnetically extracted and purified by a motorized magnetic arm and finally amplified via a duplex real-time quantitative PCR assay by a miniaturized rapid PCR module and a miniaturized fluorescence detector-all in ≤30 min. For demonstration, we use POC.auris to detect C. auris isolates from 3 major clades, with no cross reactivity against other Candida species and a limit of detection of ∼300 colony forming units per mL. Taken together, POC.auris presents a potentially useful tool for combating C. auris.

Development and Evaluation of a Fully Automated Molecular Assay Targeting the Mitochondrial Small Subunit rRNA Gene for the Detection of Pneumocystis jirovecii in Bronchoalveolar Lavage Fluid Specimens.

The fungal pathogen Pneumocystis jirovecii causes Pneumocystis pneumonia. Although the mitochondrial large subunit rRNA gene (mtLSU) is commonly used as a PCR target, a mitochondrial small subunit rRNA gene (mtSSU)-targeted MultiCode PCR assay was developed on the fully automated ARIES platform for detection of P. jirovecii in bronchoalveolar lavage fluid specimens in 2.5 hours. The assay showed a limit of detection of 800 copies/mL (approximately equal to 22 organisms/mL), with no cross-reactivity with other respiratory pathogens. Compared with the reference Pneumocystis-specific direct fluorescent antibody assay (DFA) and mtLSU-targeted PCR assay, the new assay demonstrated sensitivity of 96.9% (31/32) and specificity of 94.6% (139/147) in detecting P. jirovecii in 180 clinical bronchoalveolar lavage fluid specimens. This assay was concordant with all DFA-positive samples and all but one mtLSU PCR-positive sample, and detected eight positive samples that were negative by DFA and mtLSU PCR. Receiver operating characteristic curve analysis revealed an area under the curve of 0.98 and a threshold cycle (CT) cutoff of 39.1 with sensitivity of 90.9% and specificity of 99.3%. The detection of 39.1

Disseminated sporotrichosis following iatrogenic immunosuppression for suspected pyoderma gangrenosum.

Sporotrichosis is an infection caused by the dimorphic fungus Sporothrix schenckii and related species that often arises from traumatic inoculation of inhabited soil and organic debris into skin. The infection is usually limited to the skin in immunocompetent patients, usually as lymphocutaneous sporotrichosis. Accurate diagnosis rests on clinical data and culture, and might be facilitated by biopsy identification of suppurative and granulomatous inflammation with fungal elements. In this Grand Round, we present a dramatic case of cutaneous sporotrichosis initially presented with an atypical large ulcer without associated lymphocutaneous spread, clinically mimicking pyoderma gangrenosum, and subsequently progressed to disseminated sporotrichosis in the setting of iatrogenic immunosuppression. We further review the clinical features, risk factors, and treatment of these disseminated sporotrichosis cases, and discuss the need for improved awareness of this fungus' potential link to cause disseminated and invasive fungal infections.