Dynamics and prognostic value of plasma cell-free DNA PCR in patients with invasive aspergillosis and mucormycosis.

Aspergillus species and Mucorales agents are the primary etiologies of invasive fungal disease (IFD). Biomarkers that predict outcomes are needed to improve care. Patients diagnosed with invasive aspergillosis and mucormycosis using plasma cell-free DNA (cfDNA) PCR were retested weekly for 4 weeks. The primary outcome included all-cause mortality at 6 weeks and 6 months based on baseline cycle threshold (CT) values and results of follow-up cfDNA PCR testing. Forty-five patients with Aspergillus and 30 with invasive Mucorales infection were retested weekly for a total of 197 tests. Using the European Organization for Research and Treatment of Cancer and the Mycoses Study Group Education and Research Consortium (EORTC/MSG) criteria, 30.7% (23/75), 25.3% (19/75), and 38.7% (29/75) had proven, probable, and possible IFD, respectively. In addition, 97.3% (73/75) were immunocompromised. Baseline CT increased significantly starting at week 1 for Mucorales and week 2 for Aspergillus. Aspergillosis and mucormycosis patients with higher baseline CT (CT >40 and >35, respectively) had a nonsignificantly higher survival rate at 6 weeks, compared with patients with lower baseline CT. Mucormycosis patients with higher baseline CT had a significantly higher survival rate at 6 months. Mucormycosis, but not aspergillosis patients, with repeat positive cfDNA PCR results had a nonsignificantly lower survival rate at 6 weeks and 6 months compared with patients who reverted to negative. Aspergillosis patients with baseline serum Aspergillus galactomannan index <0.5 and <1.0 had significantly higher survival rates at 6 weeks when compared with those with index ≥0.5 and ≥1.0, respectively. Baseline plasma cfDNA PCR CT can potentially be used to prognosticate survival in patients with invasive Aspergillus and Mucorales infections. IMPORTANCE: We show that Aspergillus and Mucorales plasma cell-free DNA PCR can be used not only to noninvasively diagnose patients with invasive fungal disease but also to correlate the baseline cycle threshold with survival outcomes, thus potentially allowing the identification of patients at risk for poor outcomes, who may benefit from more targeted therapies.

Neuroinvasive Francisella tularensis Infection: Report of 2 Cases and Review of the Literature.

BACKGROUND: Neuroinvasive infection with Francisella tularensis, the causative agent of tularemia, is rare. Establishing clinical suspicion is challenging if risk factors or clinical features classically associated with tularemia are absent. Tularemia is treatable with antibiotics; however, there are limited data to inform management of potentially fatal neuroinvasive infection. METHODS: We collected epidemiologic and clinical data on 2 recent US cases of neuroinvasive F. tularensis infection, and performed a literature review of cases of neuroinvasive F. tularensis infection published after 1950. RESULTS: One patient presented with focal neurologic deficits and brain lesions; broad-range molecular testing on resected brain tissue detected F. tularensis. The other patient presented with meningeal signs; tularemia was suspected based on animal exposure, and F. tularensis grew in cerebrospinal fluid (CSF) culture. Both patients received combination antibiotic therapy and recovered from infection. Among 16 published cases, tularemia was clinically suspected in 4 cases. CSF often displayed lymphocytic pleocytosis. Among cases with available data, CSF culture was positive in 13 of 16 cases, and F. tularensis antibodies were detected in 11 of 11 cases. Treatment typically included an aminoglycoside combined with either a tetracycline or a fluoroquinolone. Outcomes were generally favorable. CONCLUSIONS: Clinicians should consider neuroinvasive F. tularensis infection in patients with meningitis and signs suggestive of tularemia or compatible exposures, lymphocyte-predominant CSF, unrevealing standard microbiologic workup, or lack of response to empiric bacterial meningitis treatment. Molecular testing, culture, and serologic testing can reveal the diagnosis. Favorable outcomes can be achieved with directed antibiotic treatment.

Accuracy of QuantiFERON in active tuberculosis suspects with comorbidities and nontuberculous mycobacterial infection in Northern California.

The QuantiFERON-TB Gold (QFT) is routinely utilized in North American health systems to detect a cellular immune response to Mycobacterium tuberculosis antigens in symptomatic and asymptomatic patients. The sensitivity of QFT in tuberculosis (TB) patients with comorbidities is not well established and the specificity of QFT in patients with nontuberculous mycobacteria (NTM) infections is incompletely understood. Between 2012 and 2023, all patients with culture-positive TB and patients with NTM infection per the expert diagnostic guidelines or biopsy-proven NTM infection who had a concurrent QFT test were included in this study. The sensitivity and specificity of QFT were measured in TB and NTM patients, respectively. In 109 patients with active TB, the overall sensitivity of QFT was 78.0% (85/109; 95% CI: 70.1, 85.7). The sensitivity was 86.0% (49/57; 95% CI: 76.6, 94.8) and 69.2% (36/52; 95% CI: 56.7, 81.8) in immunocompetent and immunocompromised patients, respectively. The overall specificity of QFT in 88 patients with NTM infection was 76.1% (67/88; 95% CI: 67.2, 85.0). After the exclusion of 17 NTM patients with risk factors for latent TB infection, the specificity was 94.4% (67/71; 95% CI: 89.1, 99.7). Two patients had NTM species known to cross-react with QFT. In two NTM patients infected with species (Mycobacterium intracellulare subsp. intracellulare and Mycobacterium intracellulare subsp. chimaera) not known to cross-react, whole genome sequencing did not detect ESAT-6 or CFP-10. In Northern California, the QFT assay demonstrated moderately low to moderately high sensitivity in TB patients and very high specificity in NTM patients, thus ruling out concerns for cross-reactivity with NTM.

Superior Accuracy of Aspergillus Plasma Cell-Free DNA Polymerase Chain Reaction Over Serum Galactomannan for the Diagnosis of Invasive Aspergillosis.

BACKGROUND: Invasive aspergillosis (IA) in immunocompromised hosts carries high morbidity and mortality. Diagnosis is often delayed because definitive diagnosis requires invasive specimen collection, while noninvasive testing with galactomannan is moderately accurate. Plasma cell-free DNA polymerase chain reaction (cfDNA PCR) represents a novel testing modality for the noninvasive diagnosis of invasive fungal disease (IFD). We directly compared the performance of Aspergillus plasma cfDNA PCR with serum galactomannan for the diagnosis of IA during routine clinical practice. METHODS: We conducted a retrospective study of all patients with suspected IFD who had Aspergillus plasma cfDNA PCR testing at Stanford Health Care from 1 September 2020 to 30 October 2022. Patients were categorized into proven, probable, possible, and no IA based on the EORTC/MSG definitions. Primary outcomes included the clinical sensitivity and specificity for Aspergillus plasma cfDNA PCR and galactomannan. RESULTS: Overall, 238 unique patients with Aspergillus plasma cfDNA PCR test results, including 63 positives and 175 nonconsecutive negatives, were included in this study. The majority were immunosuppressed (89.9%) with 22.3% 30-day all-cause mortality. The overall sensitivity and specificity of Aspergillus plasma cfDNA PCR were 86.0% (37 of 43; 95% confidence interval [CI], 72.7-95.7) and 93.1% (121 of 130; 95% CI, 87.4-96.3), respectively. The sensitivity and specificity of serum galactomannan in hematologic malignancies/stem cell transplants were 67.9% (19 of 28; 95% CI, 49.3-82.1) and 89.8% (53 of 59; 95% CI, 79.5-95.3), respectively. The sensitivity of cfDNA PCR was 93.0% (40 of 43; 95% CI, 80.9-98.5) in patients with a new diagnosis of IA. CONCLUSIONS: Aspergillus plasma cfDNA PCR represents a more sensitive alternative to serum galactomannan for noninvasive diagnosis of IA.

Mycobacterium marinum Infection after Iguana Bite in Costa Rica.

Infections after reptile bites are uncommon, and microbial etiologies are not well defined. We describe a case of Mycobacterium marinum soft-tissue infection after an iguana bite in Costa Rica that was diagnosed through 16S rRNA sequencing and mycobacterial culture. This case informs providers of potential etiologies of infection after iguana bites.

Balamuthia mandrillaris brain infection: a rare cause of a ring-enhancing central nervous system lesion. Illustrative case.

BACKGROUND: An 80-year-old man presented with subacute mental status change, dizziness, and left-sided vision loss. Magnetic resonance imaging demonstrated a ring-enhancing right parietooccipital lesion. OBSERVATIONS: Biopsy and laboratory testing demonstrated an amoebic Balamuthia mandrillaris infection. Fewer than 200 cases of this infection have been recognized in the United States, and no standardized treatment regimen currently exists. LESSONS: Rapid antimicrobial therapy with miltefosine, azithromycin, fluconazole, flucytosine, sulfadiazine, and albendazole was initiated. The pathophysiology, diagnosis, and management of this infection and the patient's course were reviewed. The importance of biopsy for pathologic and laboratory diagnosis and rapid treatment initiation with a multidisciplinary team was reinforced.

Clinical accuracy of malaria loop-mediated isothermal amplification assay as a stand-alone screening tool at a non-endemic Northern California regional health system.

Malaria is critical to rule out in febrile returned travelers. We evaluated the performance of the Alethia Malaria loop-mediated isothermal amplification (LAMP) assay for rapid one-time malaria screening using nucleic acid amplification testing. Retrospective analysis of 51 archival blood specimens collected from 32 patients with malaria and 25 uninfected controls showed Malaria LAMP assay to have sensitivity of 100% (95% CI 93.0-100) and specificity of 100% (95% CI 86.7-100) using blood smear microscopy as the reference standard. Prospective evaluation of Malaria LAMP as a single screening test in 205 patients identified 4 (1.95%) positives which were all confirmed as Plasmodium falciparum by PCR, with parasitemia quantified as <0.1% (n = 2), 1.0% (n = 1), and 4.6% (n = 1). Alternative diagnoses were found in 129 of 201 (64.2%) of LAMP-negative patients, and no patient was subsequently diagnosed with malaria. The Malaria LAMP offers a sensitive and rapid stand-alone screening alternative in non-endemic settings.

Accuracy of Pneumocystis jirovecii Plasma Cell-Free DNA PCR for Noninvasive Diagnosis of Pneumocystis Pneumonia.

Pneumocystis pneumonia (PCP) caused by Pneumocystis jirovecii is a serious infection in immunocompromised hosts which requires prompt diagnosis and treatment. The recommended specimen for diagnosis of PCP is bronchoalveolar lavage (BAL) fluid, which is invasive and may not be possible in unstable patients. The aim of this study was to evaluate the accuracy of noninvasive P. jirovecii plasma cell-free DNA (cfDNA) PCR using recently optimized preanalytical and analytical methods. Adult patients undergoing clinical testing for PCP with direct fluorescent antibody stain (DFA), respiratory PCR, and/or ß-d-glucan were included in this study. Sensitivity and specificity P. jirovecii plasma cfDNA PCR was determined in PCP suspects categorized as proven and probable. A total of 149 patients were included in this study, of which 10 had proven and 27 had probable PCP. Most patients (95.9%, 143/149) were immunocompromised, including hematological malignancies (30.1%), bone marrow transplant (11.2%), solid organ transplantation (47.6%), and HIV/AIDS (4.2%). P. jirovecii plasma cfDNA PCR showed sensitivity and specificity of 100% (10/10; 95% confidence interval [CI], 69.2 to 100) and 93.4% (127/136; 95% CI, 87.8 to 96.9), and 48.6% (18/37; 95% CI, 31.9 to 65.6) and 99.1% (108/109; 95% CI, 94.9 to 100) in proven and proven/probable cases, respectively. P. jirovecii cell-free DNA PCR was similar in sensitivity but with substantially improved specificity over ß-d-glucan (sensitivity, 60.0% [18/30; 95% CI, 40.6 to 77.3]); specificity, 66.7% [22/33; 95% CI, 48.2 to 82.0]) in patients with proven/probable PCP. Plasma cfDNA PCR offers a noninvasive testing option for early and accurate diagnosis of PCP, particularly in patients who cannot tolerate bronchoscopy.

Clinical Accuracy and Impact of Plasma Cell-Free DNA Fungal Polymerase Chain Reaction Panel for Noninvasive Diagnosis of Fungal Infection.

BACKGROUND: Invasive fungal infection (IFI) is a growing cause of morbidity and mortality in oncology and transplant patients. Diagnosis of IFI is often delayed due to need for invasive biopsy and low sensitivity of conventional diagnostic methods. Fungal cell-free DNA (cfDNA) detection in plasma is a novel testing modality for the noninvasive diagnosis of IFI. METHODS: A novel bioinformatic pipeline was created to interrogate fungal genomes and identify multicopy sequences for cfDNA polymerase chain reaction (PCR) targeting. A real-time PCR panel was developed for 12 genera and species most commonly causing IFI. Sensitivity and specificity of the fungal PCR panel were determined using plasma samples from patients with IFI and non-IFI controls. Clinical impact of the fungal PCR panel was evaluated prospectively based on the treating team's interpretation of the results. RESULTS: Overall, the sensitivity and specificity were 56.5% (65/115; 95% confidence interval [CI], 47.4-65.2) and 99.5% (2064/2075; 95% CI, 99.0-99.7), respectively. In the subset of patients with an optimized plasma volume (2 mL), sensitivity was 69.6% (48/69; 95% CI, 57.9-79.2). Sensitivity was 91.7% (11/12; 95% CI, 62.5-100) for detection of Mucorales agents, 56.3% (9/16; 95% CI, 33.2-76.9) for Aspergillus species, and 84.6% (11/13; 95% CI, 56.5-96.9) for Candida albicans. In a prospective evaluation of 226 patients with suspected IFI, cfDNA testing was positive in 47 (20.8%) patients and resulted in a positive impact on clinical management in 20 of 47 (42.6%). CONCLUSIONS: The fungal cfDNA PCR panel offers a noninvasive approach to early diagnosis of IFI, providing actionable results for personalized care.

Reactivation of Chagas Disease in a Patient With an Autoimmune Rheumatic Disease: Case Report and Review of the Literature.

Reactivation of Chagas disease has been described in immunosuppressed patients, but there is a paucity of literature describing reactivation in patients on immunosuppressive therapies for the treatment of autoimmune rheumatic diseases. We describe a case of Chagas disease reactivation in a woman taking azathioprine and prednisone for limited cutaneous systemic sclerosis (lcSSc). Reactivation manifested as indurated and erythematous cutaneous nodules. Sequencing of a skin biopsy specimen confirmed the diagnosis of Chagas disease. She was treated with benznidazole with clinical improvement in the cutaneous lesions. However, her clinical course was complicated and included disseminated CMV disease and subsequent septic shock due to bacteremia. Our case and review of the literature highlight that screening for Chagas disease should be strongly considered for patients who will undergo immunosuppression for treatment of autoimmune disease if epidemiologically indicated.

Utilization, Yield, and Accuracy of the FilmArray Meningitis/Encephalitis Panel with Diagnostic Stewardship and Testing Algorithm.

The impact of diagnostic stewardship and testing algorithms on the utilization and performance of the FilmArray meningitis/encephalitis (ME) panel has received limited investigation. We performed a retrospective single-center cohort study assessing all individuals with suspected ME between February 2017 and April 2019 for whom the ME panel was ordered. Testing was restricted to patients with cerebrospinal fluid (CSF) pleocytosis. Positive ME panel results were confirmed before reporting through correlation with direct staining (Gram and calcofluor white) and CSF cryptococcal antigen or by repeat ME panel testing. Outcomes included the ME panel test utilization rate, negative predictive value of nonpleocytic CSF samples, test yield and false-positivity rate, and time to appropriate deescalation of acyclovir. Restricting testing to pleocytic CSF samples reduced ME panel utilization by 42.7% (263 versus 459 tests performed) and increased the test yield by 61.8% (18.6% versus 11.5% positivity rate; P < 0.01) with the application of criteria. The negative predictive values of a normal CSF white blood cell (WBC) count for ME panel targets were 100% (195/195) for nonviral targets and 98.0% (192/196) overall. All pathogens detected in nonpleocytic CSF samples were herpesviruses. The application of a selective testing algorithm based on repeat testing of nonviral targets avoided 75% (3/4) of false-positive results without generating false-negative results. The introduction of the ME panel reduced the duration of acyclovir treatment from an average of 66 h (standard deviation [SD], 43 h) to 46 h (SD, 36 h) (P = 0.03). The implementation of the ME panel with restriction criteria and a selective testing algorithm for nonviral targets optimizes its utilization, yield, and accuracy.

Reproducibility of positive results for rare pathogens on the FilmArray GI Panel.

Though the FilmArray GI Panel has a reported aggregate specificity and reproducibility of >97% and > 99%, respectively, the reproducibility is less understood in clinical practice. We measured the reproducibility of positive results for low-prevalence pathogens. Samples with positive results for selected targets were repeated using a different FilmArray module. Overall, 331 of 373 (89%) results were reproducible. Giardia lamblia (57/57, 100%), Cryptosporidium spp. (61/63, 97%), Cyclospora cayetanensis (34/35, 97%), Plesiomonas shigelloides (17/18, 94%), and Rotavirus A (76/77, 99%) were highly reproducible, while Adenovirus F40/41 (38/54, 70%), Vibrio spp. (8/10, 80%), V. cholerae (3/8, 37.5%), and Yersinia enterocolitica (36/50, 72%) were poorly reproducible. Review of 38 patients with nonreproducible results showed that 19 (50%) had evidence of gastroenteritis and only 6 (16%) had possible infection with the organism that showed a nonreproducible result. Higher false-positive rates with certain targets on FAGP emphasize the need for diagnostic stewardship.